Patent Grant
7341836
The present invention provides modified cyclic nucleotide gated (CNG) channels. In particularly preferred embodiments, the modified CNG channels exhibit increased sensitivity and specificity for cAMP, as compared to wild-type CNG channels. In additional embodiments, regulation by Ca2+-calmodulin has been removed in the modified CNG channels. Convenient optical methods for detecting changes in cAMP, taking advantage of the Ca2+ permeability of the channel are also provided by the present invention. In addition, electrophysiological methods are further provided.
Cyclic AMP (cAMP) is a ubiquitous intracellular second messenger that coordinates diverse cellular functions. It is produced in response to a large variety of extracellular stimuli, including hormones and neurotransmitters. Typically, these agents bind to receptors on the extracellular surface, the receptors activate G-proteins on the intracellular surface and the G-proteins in turn activate adenylyl cyclase, the enzyme that produces cAMP.
cAMP signals are considered to be complex, as evidenced by cAMP's differential regulation of over 200 cellular targets. In addition, the enzymes involved in cAMP metabolism are known to be regulated by numerous other signaling pathways. Unfortunately, as discussed further below, an understanding of cAMP signals has been elusive, due to the fact that current methods for measuring cAMP lack both temporal and spatial resolution. Thus, what is needed are high-resolution means that provided the requisite resolution in order to measure intracellular cAMP.
For example, the standard method for measuring cAMP accumulation within cells is to treat cells with [3H]-adenine to label the ATP pool, and then measure the conversion of [3H]ATP to [3H]cAMP at different time points (See e.g., Evans et al., Mol. Pharmacol., 26:395-404 [1984]). This method is typically done on hundreds of thousands to millions of cells. As a consequence, the method has no spatial resolution and it cannot be used to assess cell-to-cell variability within a population. In addition, it is labor-intensive because cAMP accumulation can be measured only at discrete time points (i.e., in contrast to fluorescence or electrophysiological techniques, it does not provide a continuous readout). This is an important consideration in screening applications. In addition, the method has low temporal resolution, as it is impractical to measure cAMP accumulation in less than 5 second increments. Thus, it is likely that rapid changes in cAMP will be missed when this technique is used.
A second method currently used in the art involves measuring the changes in fluorescence energy transfer between labeled subunits of cAMP-dependent protein kinase, which dissociate upon binding of cAMP. Fluorescent subunits are either prepared biochemically and microinjected (See, Adams et al., Nature 349:694-697 [1991]), or are genetically encoded (See, Zaccolo et al., Nat. Cell. Biol., 2:25-29 [2000]). Although this method does allow for detection of cAMP changes in single cells, it has very low spatial resolution due to limitations in the wavelength utilized (i.e., it is limited by the wavelengths of visible light; 400-800 nm). It also has low temporal resolution, due to the slow reassociation of labeled subunits (i.e., a half-time of 100-200 seconds at typical subunit concentrations; See, Ogreid and Doskeland, Biochem., 22:1686-1696 [1983]), and the tendency of catalytic subunits that catalyze phosphorylation to accumulate in the nucleus (See, Harootunian et al., Mol. Cell. Biol., 4:993-1002 [1993]). In addition, in order to overwhelm endogenous kinase, several micromolar labeled subunits are usually introduced into the test system. This strongly buffers natural cAMP signals, and causes functional alterations of cellular targets due to extensive phosphorylation. Thus, there remains a need in the art for methods and compositions that further elucidate the activities of receptors, G-proteins, phosphodiesterases (PDEs), adenylyl cyclases, and other proteins important in cAMP signalling.
The present invention provides modified CNG channels, in which the sensitivity and specificity for cAMP are increased. In addition, regulation by Ca2+-CaM is removed in the modified CNG channels. Convenient optical methods for detecting changes in cAMP, taking advantage of the Ca2+ permeability of the channel are also provided by the present invention.
In particular, the present invention provides isolated nucleic acids encoding a modified olfactory cyclic nucleotide-gated ion channel, wherein the channel comprises mutations which together impart increased cAMP sensitivity, decreased cGMP sensitivity, decreased nitric oxide sensitivity and decreased calcium-calmodulin sensitivity.
The present invention also provides isolated nucleic acids encoding a modified olfactory cyclic nucleotide-gated ion channel, in which the channel comprises at least one mutation selected from the group consisting of a C460W mutation and a E583M mutation. In some embodiments, the channel comprises a E583M mutation; a C460W mutation and a E583M mutation; or a 61-90 deletion, a C460W mutation, and a E583M mutation. In a subset of these embodiments, the channel comprises an amino acid sequence selected from the group consisting of SEQ ID NO:5, SEQ ID NO:6, and SEQ ID NO:7. Also provided in some embodiments are polypeptides encoded by the isolated nucleic acids, and expression vectors comprising the isolated nucleic acids. In preferred embodiments, the vector is a recombinant adenovirus vector. Furthermore, the invention provides host cells comprising the expression vectors, in which the host cell is selected from the group consisting of a prokaryotic cell and a eukaryotic cell. In preferred embodiments, the eukaryotic cell is selected from the group consisting of a human embryonic kidney-293 cell, a GH4C1 pituitary cell, and a rat C6-2B glioma cell.
The present invention also provides methods for determining local intracellular cAMP concentration within an eukaryotic cell, comprising the steps of providing: at least one eukaryotic cell, and a nucleic acid encoding a modified olfactory cyclic nucleotide-gated ion channel, in which the channel comprises at least one mutation selected from the group consisting of a C460W mutation and a E583M mutation; and contacting the eukaryotic cell with the nucleic acid under conditions suitable for expressing the modified olfactory cyclic nucleotide-gated ion channel in the eukaryotic cell; measuring intracellular calcium concentration in the eukaryotic cell; and determining local intracellular cAMP concentration based upon the intracellular calcium concentration. In some embodiments, the contacting step comprises infecting the eukaryotic cell with a recombinant adenovirus comprising the nucleic acid. In some preferred embodiments, the measuring step comprises contacting the eukaryotic cell with a stimulus. In a subset of these embodiments, the stimulus is selected from the group consisting of an adenylate cyclase activator, a G-protein activator, and a phosphodiesterase inhibitor. In particularly preferred embodiments, the measuring step comprises monitoring calcium flux with a fluorescent calcium indicator. Appropriate fluorescent calcium indicators include but are not limited to fura-2, indo-1, quin-2, fluo-3 and rhod-2.
Moreover, the present invention provides methods for determining local intracellular cAMP concentration within an eukaryotic cell, comprising the steps of providing: at least one eukaryotic cell, and a nucleic acid encoding a modified olfactory cyclic nucleotide-gated ion channel, wherein the channel comprises at least one mutation selected from the group consisting of a C460W mutation and a E583M mutation; and contacting the eukaryotic cell with the nucleic acid under conditions suitable for expressing the modified olfactory cyclic nucleotide-gated ion channel in the eukaryotic cell; measuring the electric current across the plasma membrane of the eukaryotic cell; and determining local intracellular cAMP concentration based upon the electric current. In some embodiments, contacting step comprises infecting the eukaryotic cell with a recombinant adenovirus comprising the nucleic acid. In some preferred embodiments, the measuring step comprises contacting the eukaryotic cell with a stimulus. In a subset of these embodiments, the stimulus is selected from the group consisting of an adenylate cyclase activator, a G-protein activator, and a phosphodiesterase inhibitor. In some embodiments, the measuring step comprises monitoring the electric current with a patch-clamp technique selected from the group consisting of perforated patch-clamp technique and a whole-cell patch-clamp technique. In related embodiments, the determining step comprises calibrating the electric current with respect to cAMP concentration by obtaining a cAMP-dose response curve for the modified cyclic nucleotide-gated ion channel with an inside-outside patch clamp technique.
Also provided by the present invention are methods for determining whether a candidate compound is capable of modulating local intracellular cAMP concentration within a eukaryotic cell, comprising the steps of providing: at least one eukaryotic cell expressing a modified olfactory cyclic nucleotide-gated ion channel, wherein the channel comprises at least one mutation selected from the group consisting of a C460W mutation and a E583M mutation, and a drug candidate; and determining local intracellular cAMP concentration within the eukaryotic cell in the presence and absence of the drug candidate. In some embodiments, the expressing of the modified olfactory cyclic nucleotide-gated ion channel is accomplished by infection of the eukaryotic cell with a recombinant adenovirus. In some preferred embodiments, the determining of the local intracellular cAMP concentration is accomplished by measuring intracellular calcium concentration in the eukaryotic cell in the presence and absence of the drug candidate. In other preferred embodiments, the determining of the local intracellular cAMP concentration is accomplished by measuring the electric current the plasma membrane of the eukaryotic cell in the presence and absence of the drug candidate.
The present invention provides modified cyclic nucleotide gated (CNG) channels. In particularly preferred embodiments, the modified CNG channels exhibit increased sensitivity and specificity for cAMP, as compared to wild-type CNG channels. In additional embodiments, regulation by Ca2+-calmodulin has been removed in the modified CNG channels. Convenient optical methods for detecting changes in cAMP, taking advantage of the Ca2+ permeability of the channel are also provided by the present invention. In addition, electrophysiological methods are further provided.
In some preferred embodiments, the present invention provides genetically-modified cyclic nucleotide-gated ion channels. This class of channels is directly opened by the binding of cyclic nucleotides. These channels were discovered in retinal photoreceptor cells and olfactory receptor neurons, where they generate the electrical response to light and odorants. The native retinal channel is cyclic GMP (cGMP) specific, while the native olfactory channel is equally sensitive to cAMP and cGMP. Native channels consist of both and subunits, both of which bind cyclic nucleotides. Open channels allow cations (Na+, K+, Ca2+, Mg2+) to pass through the cell membrane. Thus, the activation of channels is readily detected with both electrophysiological and Ca2+-imaging techniques, as known in the art.
As discussed in greater detail herein, cyclic nucleotide-gated (CNG) channels have several properties that make them attractive as cAMP sensors. First, they incorporate into discrete regions of the surface membrane where adenylyl cyclase is known to reside. Second, several hundred channels in a cell give a robust signal without significantly buffering the cAMP being measured. Third, cyclic nucleotide-gated channels respond rapidly to changes in cyclic nucleotide concentration. Fourth, the sensor can be calibrated in different cell types by measuring the apparent cAMP affinity in excised membrane patches. This controls for the possible modulation of CNG channels by endogenous proteins.
Retinal and olfactory α subunits form functional channels on their own, and the rat olfactory α subunit was previously used to measure cAMP accumulation under strong stimulus conditions. However, the wild-type α subunit has several drawbacks for measuring physiologic cAMP signals. First, wild-type channels (See,
In some embodiments of the present invention, a mutant rat olfactory channel α subunit is used. In order to create the mutant, modifications were made to the rat olfactory channel α subunit to increase its sensitivity and specificity for cAMP and to remove regulation by Ca2+−CaM. In particular, new mutations, designated as “C460W” and “E583M” were used to produce a channel with significantly increased sensitivity and specificity for cAMP. The resultant C460W/E583M channel has K1/2 values of 1.2 and 12 μM for cAMP and cGMP (See, Table 1, below). To remove regulation of the channel by Ca2+-CaM binding residues 61 through 90 were deleted.
The Δ61-90/C460W/E583M channel has two important advantages. First, it is virtually insensitive to cGMP. The current elicited by a saturating concentration of cGMP is 16% of the current elicited by saturating cAMP (See, Table 1, below). In addition, 36 μM cGMP is required to elicit half of that tiny response. Second, the channel is not inhibited by Ca2+-CaM. The sensitivity to cAMP is reduced (K1/2=14.5 μM), which allows cAMP to be measured in the upper end of the physiologic range. The use of both mutant channels allows cAMP to be measured over the entire physiologic range (0.1 to 50 μM).
As described in greater detail herein, the effects of modifications to CNG channels were assessed using the Hill equation, I/Imax=[cNMP]N/([cNMP]N+K1/2N), where I/Imax is the fraction of maximal current, cNMP represents cyclic nucleotide, K1/2 is the concentration that gives half-maximal current, and N is the Hill coefficient, an index of cooperativity. I/ImaxcGMP/ImaxcAMP is the current induced by saturating cGMP divided by the current induced by saturating cAMP. Data (mean±standard deviation of three experiments) were obtained from excised membrane patches using standard patch clamp techniques.
In particularly preferred embodiments, the coding sequence of the channels have been incorporated into adenovirus constructs, which allow for efficient expression in a variety of cell types. The adenovirus constructs of preferred embodiments of the present invention are deleted for the E3 region and have the E1 region replaced by the CNG channel alleles under the control of the cytomegalovirus major intermediate promoter. The channels have been used to measure cAMP in two different assay designs.
One assay system involves a convenient, optical method for detecting changes in cAMP, which takes advantage of the Ca2+ permeability of the channel. There are several advantages of measuring Ca2+ influx through CNG channels as a way to monitor changes in cAMP. Importantly, this assay is very simple to implement in either cell populations or single cells. In addition, the measurement has a high signal-to-noise ratio. Furthermore, it is sensitive to small changes in adenylyl cyclase activity. In contrast to the present invention, in traditional assays, strong stimuli and phosphodiesterase (PDE) inhibitors are often required to detect cAMP accumulation. Natural cAMP signals are severely distorted by the necessity to inhibit PDE, the enzyme responsible for degrading cAMP. In contrast to the currently used means, the novel channels described herein readily detect changes in cAMP in response to physiologic stimuli, and in the absence of PDE inhibitors. This assay is particularly well-suited as an initial screen for changes in cAMP.
Another assay system that finds use with the present invention involves the use of electrophysiological assays, in which either perforated patch or whole-cell configurations of the patch clamp technique are used to measure currents through the ion channels. This assay is particularly appropriate when more precise measurements of local cAMP are required. These measurements take advantage of the fast response of CNG channels to changes in cAMP concentration, without the distortions that arise from the Ca2+ handling properties of the cell. The higher dynamic range, increased temporal resolution, and more accurate measurement of cAMP concentration provided by these methods find use in providing information regarding cAMP signals and signalling systems. The increased sensitivity of the novel CNG channels of the present invention were confirmed using in vivo assays by monitoring cAMP-induced Ca2+ influx through the channels in cell populations and by measuring cAMP-induced currents in the whole-cell patch clamp configuration.
Further, as discussed herein, the present invention provides methods and compositions for measuring changes in intracellular cAMP. A very large number of natural agents (e.g., hormones, neurotransmitters, odorants) and drugs bind to G-protein coupled receptors and cause changes in cAMP levels. As the present invention provides a system with demonstrated robust expression of the adenovirus-encoded sensor in several cell types, the present invention provides means for both high-throughput pharmacological screens as well as for many research purposes.
Indeed, the compositions of the present invention have been used: 1) to demonstrate the existence of distinct cAMP signals in different regions of a simple cell; 2) to probe the interplay between stimulatory and inhibitory G-protein regulation of adenylyl cyclase; 3) to identify the phosphodiesterase (PDE) subtypes that shape cAMP levels in non-excitable and excitable cells (these experiments also provided the inhibition constants of several PDE inhibitors in the intact cell). The present invention provides sensors that have a high signal-to-noise ratio over most of the physiologic range of cAMP concentrations (100 nM to 100 μM). However, it is contemplated that additional sensors of the present invention will find use over the entire range of physiological cAMP concentrations (i.e., down to 20 nM).
As discussed in greater detail elsewhere herein, in some particularly preferred embodiments, the present invention provides compositions comprising several modifications to the WT olfactory CNG channel that significantly enhance its utility as a cAMP sensor. Testing of three novel constructs is described herein. As discussed in greater detail herein, in comparison with WT CNG channels, the E583M channel (SEQ ID NO:5) is more sensitive to cAMP and less sensitive to cGMP. However, two additional mutations produced channels that make even better cAMP sensors. The C460W/E583M channel (SEQ ID NO:6) has a high affinity for cAMP, allowing it to detect cAMP at levels which activate PKA. The Δ61-90/C460W/E583M channel (SEQ ID NO:7) is almost as sensitive to cAMP as the E583M channel. However, the Δ61-90/C460W/E583M channel has two important advantages: it is not inhibited by Ca2+-CaM, and it is barely activated by cGMP. Thus, the mutant CNG channels provided by the present invention provide the first ion channels that have been tailored for measurement of cAMP.
The present invention also provides convenient assay systems for detecting changes in local cAMP concentrations and examining PDE activities. PDEs, the crucial terminators of cAMP and cGMP signals, were discovered almost 40 years ago (Drummond and Perrot-Yee, J. Biol. Chem., 236:1126-1129 [1961]). Since then, PDEs have been classified into eleven families according to substrate specificity, regulation, pharmacology, and more recently, amino acid homology (Beavo, Adv. Second Messenger Phosphoprot. Res., 22:1-38 [1988]; Beavo, Physiol. Rev., 75:725-748 [1995]; Conti et al., Endocrine Res., 16:370-389 [1995]). Studies have confirmed differential regulation of PDE families by Ca2+-calmodulin (Ca2+-CaM), G-proteins, phosphorylation, and cyclic nucleotides. The diversity of PDE families has led to the realization that PDE activity is a central element in the control of second messenger signaling, as important as adenylyl and guanylyl cyclase activity in shaping cyclic nucleotide signals. Yet, little is known about how PDE regulates cyclic nucleotide signals in vivo or how cyclic nucleotide signals differentially regulate hundreds of cellular targets because, outside of specialized cells, there are few convenient real-time measures of cyclic nucleotide concentration. However, the present invention provides the means to address these questions and obtain real-time measurements of cyclic nucleotide concentrations.
In some particularly preferred embodiments, both WT and modified CNG channels are used. There are several advantages provided by the methods and compositions of the present invention involving measuring Ca2+ influx through CNG channels as a way to monitor changes in cAMP. First, this assay is very simple to implement in either cell populations or single cells. Second, the measurement has a high signal-to-noise ratio. Third, it is sensitive to small changes in AC activity. This is due, in part, to the fact that CNG channels detect local rather than total cellular cAMP. With traditional assays, high agonist and PDE inhibitor concentrations are often required to detect cAMP accumulation. In contrast, the channels provided by the present invention readily detect changes in cAMP caused by subsaturating agonist concentration in the absence of PDE inhibitors. In addition to being easy to use, the present invention provides improved spatial and temporal resolution, as well as means for calibration to provide precise measures of cAMP concentrations. Thus, among other utilities, the assay systems presented herein find use as initial screens for changes in cAMP in response to physiological stimuli and/or pharmacological agents.
The present invention provides significant improvements in assay systems. However, in some cases, it is difficult to extract the precise cAMP concentration from these assay systems because the responses cannot be calibrated (i.e., with known intracellular cAMP), and because Ca2+ handling and depletion of free fura-2 become issues at high Ca2+ influx rates. Nonetheless, any of these limitations can be overcome when more precise measurements of local cAMP are required, by determining the cAMP concentration using electrophysiological methods as known in the art (Rich et al. [2000], supra).
During the development of the present invention, observations were made using HEK-293 and C6-2B glioma cells, as well as GH4C1, that CNG channels measure cAMP produced in subcellular compartments or microdomains near the plasma membrane, and that diffusion of cAMP between the microdomains and the bulk cytosol is severely hindered (See, Rich et al. [2000], supra). This conclusion was based on several lines of evidence. First, forskolin-induced increases in cAMP concentration measured using CNG channels (>25 μM) were much greater than the increases in cAMP concentration averaged throughout the accessible cell volume (1-2 μM). Second, forskolin-induced cAMP accumulation was easily detected by CNG channels in the rapidly-dialyzed, whole-cell patch clamp configuration. Third, the wash-in of cAMP from the patch pipette to the CNG channels was much slower than would be expected based upon the rapid exchange of the bulk cytosol. All of these results were described by a three-compartment model (microdomain, cytosol, whole-cell pipette) in which the transfer rates between compartments were determined using data from the wash-in experiments.
During the development of the present invention, much was learned about the two dimensional localization of proteins involved in cellular signaling. For example, certain isoforms of PDE are differentially distributed between particulate (membrane-localized) and supernatant (cytosolic) cellular fractions. In addition, PDE kinetics may vary with cellular localization (See, Bolger et al., Biochem. J., 328:539-548 [1997]). Elements of G-protein signaling pathways have been shown to preferentially localize within caveolae (i.e., distinct regions of the plasma membrane; See e.g., Rybin et al., J. Biol. Chem., 275:41447-41457 [2000]). Furthermore, a recent report has described the localization of different voltage-gated K+ channels to distinct populations of lipid rafts (Martens et al., J. Biol. Chem., 276:8409-8414 [2001]).
However, two dimensional localization within the plasma membrane does not account for the three dimensional compartmentalization of cAMP signals. Data obtained during the development of the present invention show that proximity to AC does not provide sufficient cAMP concentrations for activation of effector proteins because, in essence, each cAMP molecule diffuses away more rapidly than the next molecule is synthesized. Thus, PDE activity alone is unlikely to be responsible for compartmentalized cAMP signals, because PDE activity only lowers cAMP levels. Thus, it is contemplated that PDEs regulating cAMP levels detected by CNG channels are localized within the same three dimensional compartment as AC and CNG channels. Importantly, by regulating cAMP concentration within this compartment, PDE activity affects the rate of cAMP flux between compartments. This PDE is contemplated to be a component of the particulate PDE fractions. In addition, cytosolic PDE is contemplated to regulate cAMP in other cellular compartments.
During the development of the present invention, Ca2+ influx through CNG channels was used to detect changes in cAMP concentration in both nonexcitable HEK-293 cells and excitable pituitary GH4C1 cells. One goal was to assess the PDE subtypes responsible for shaping cAMP signals near the plasma membrane. In order to study the effects of PDEs, it is important to be able to first detect cAMP changes in the absence of PDE inhibitors. There was no difficulty with this in either cell type, despite the modest levels of cAMP accumulation in response to forskolin or VIP stimulation reported previously (Mollard et al., Biochem. J., 284:637-640 [1992]; Fagan et al., J. Biol. Chem., 271:12438-12444 [1996]; and Fagan et al., J. Biol. Chem., 275:40187-40194 [2000]). In HEK-293 cells, only nonselective and PDE-type-IV selective inhibitors decreased PDE activity. The in vivo estimates of KI, for three PDE inhibitors (IBMX, RO-20-1724, and rolipram) described herein, are consistent with IC50 values estimated in vitro for PDE type IV. These data are also consistent with a previous report that identified two isoforms of PDE type IV (PDE4D3 and PDE4D5) in HEK-293 cells based on Western blot analysis (Hoffmann et al., EMBO J., 18:893-903 [1999]). Inhibition of PDE activity did not reveal basal AC activity in HEK-293 cells. In GH4C1 cells, inhibitors selective to PDE types I and IV decreased PDE activity. Using the high cAMP affinity CNG channels, inhibition of PDE type I activity led to a rise in local cAMP concentration in the absence of AC stimulation. This increase revealed substantial basal AC activity in GH4C1 cells. Under the same conditions, no change in cAMP level was observed when PDE type IV inhibitors were added. However, using the lower cAMP affinity, Δ61-90/C460W/E583M channels, inhibition of either PDE type I or type IV increased forskolin-induced cAMP accumulation. These observations point to the presence of two different PDE types: a low Km, PDE type I, and a higher Km, PDE type IV. Based on the apparent cAMP affinities of the two channel constructs, the two Km values were estimated to be <1 μM and >5 μM.
Interestingly, the data in GH4C1 cells indicate the presence of a futile cycle of cAMP synthesis and hydrolysis. Restricting this phenomenon to subcellular compartments would be advantageous energetically, but the question remains as to why the additional energy is expended by the cells. One possibility is that constant AC and PDE activity allow the system to respond rapidly to a stimulus. In essence, the enzymes are poised to respond to changes in active G-protein or changes in internal Ca2+, allowing for rapid increases or decreases in cAMP levels. A similar situation exists in light-adapted photoreceptor cells (See e.g., Nikonov et al., J. Gen. Physiol., 116:795-824 [2000]). Nonetheless, an understanding of the mechanism(s) is not necessary in order to use the present invention.
The existence of PDE type I in GH4C1 cells is consistent with observations showing that CaM antagonists inhibit the hydrolysis of cAMP (Sletholt et al., Acta. Physiol. Scand., 130:333-343 [1987]). Furthermore, a high Km (28.6 μM) and a low Km (0.66 μM) PDE have been identified in both GH3 and GH4C1 cells, each PDE with a different IC50 for theophylline (Gautvik et al., Mol. Cell. Endocrinol., 26:295-308 [1982]). These Km values are consistent with the in vivo estimates of the Km values for PDEs that regulate local cAMP levels discussed herein. The presence of both high and low Km PDEs offers two possibilities for the shaping of cAMP signals: (1) that both PDE types regulate cAMP levels within a single compartment, or (2) that they differentially regulate cAMP levels in different cellular compartments.
If the two PDE types coexist in the same subcellular compartment then the low Km PDE would be likely to regulate cAMP levels under basal conditions. Upon further activation of AC, this PDE would be overwhelmed, and large concentrations of cAMP would accumulate locally. The high Km PDE would be able to efficiently hydrolyze cAMP at these elevated concentrations. Furthermore, it is contemplated that these PDE types are differentially regulated, allowing different feedback mechanisms to control cAMP levels within this compartment.
If the activity of the two PDE types is compartmentalized, it is contemplated that distinct cyclic nucleotide signals are capable of occurring simultaneously in different regions of the cell. Indeed, this possibility seems likely because cAMP can be excluded from areas of the cell by PDE activity (See, Jurevicius and Fischmeister, Proc. Natl. Acad. Sci. USA 93:295-299 [1996]), cAMP is produced in microdomains with restricted diffusional access to the bulk cytosol and different pools of cGMP have different functional effects in ECV304 epithelial cells (Zolle et al., J. Biol. Chem., 275:25892-25899 [2000]).
It is contemplated that the present invention will find use in measuring local cAMP signals in response to a variety of extracellular stimuli. Indeed, the present invention provides means to investigate how cAMP is capable of differentially regulating more than 200 cellular targets. This requires a quantitative knowledge of how cAMP signals are initiated and terminated. In addition, different PDE subtypes are likely to play crucial roles in shaping the signals in different cellular compartments. Within these compartments, cAMP levels are likely to vary dynamically (Brooker, Science 182:933-934 [1973]; Cooper et al., Nature 374:421-424 [1995]; and Cooper et al., Adv. Second Messenger Phosphoprotein Res., 32:23-51 [1998]), particularly in excitable cells, where Ca2+ levels oscillate during a train of action potentials. For example, it is contemplated that modulation of PDE type I by Ca2+-CaM in excitable GH4C1 cells contributes to transient or oscillating cAMP signals. In general, dynamic cAMP signals would escape detection with conventional techniques.
The present invention provides modified cyclic nucleotide gated (CNG) channels. In particularly preferred embodiments, the modified CNG channels exhibit increased sensitivity and specificity for cAMP, as compared to wild-type CNG channels. In additional embodiments, regulation by Ca2+-calmodulin has been removed in the modified CNG channels. Convenient optical methods for detecting changes in cAMP, taking advantage of the Ca2+ permeability of the channel are also provided by the present invention. In addition, electrophysiological methods are further provided.
Retinal rod outer segments are the most closely examined second messenger signaling system, in part because the endogenous cyclic nucleotide-gated (CNG) channels have been used as real-time detectors of cGMP concentration (Baylor et al., J. Physiol., 288:589-611 [1979]). Information from biochemical measurements of guanylyl cyclase and cGMP-specific PDE have been combined with real-time measurements of cGMP concentration to characterize the relationship between these enzymes in regulating cGMP levels (See e.g., Fung et al., Proc. Natl. Acad. Sci. USA 78:152-156 [1981]; Koch and Stryer, Nature 334:64-66 [1988]; Gorczyca et al., Proc. Natl. Acad. Sci. USA 91:4014-4018 [1994]; Koutalos et al., J. Gen. Physiol., 106:891-921 [1995a]; Koutalos et al., J. Gen. Physiol., 106:863-890 [1995b]; He et al., Neuron 20:95-102 [1998b]; Tsang et al., Science 282:117-121 [1998]; Chen et al., Nature 403:557-560 [2000]; Leskov et al., Neuron 27:525-537 [2000]; and Nikonov et al., J. Gen. Physiol., 116:795-824 [2000]). The convergence of different approaches has led to an unprecedented understanding of feedback signaling within this model system (See e.g., Stryer, J. Biol. Chem., 266:10711-10714 [1991]; Lagnado and Baylor, Neuron 8:995-1002 [1992]; Pugh and Lamb, Biochim. Biophys. Acta 1141:111-149 [1993]; Yarfitz and Hurley, J. Biol. Chem., 269:14329-14332 [1994]; Yau, Invest. Ophthalmol. Vis. Sci., 35:9-32 [1994]; Polans et al., Trends Neurosci., 19:547-554 [1996]; Pugh et al., Biosci. Rep., 17:429-473 [1997]; and Molday, Invest. Ophthalmol. Vis. Sci., 39:2493-2513 [1998], for review). Nonetheless, an understanding of the mechanism(s) is not necessary in order to use the present invention.
During the development of the present invention, it was determined that cAMP concentration can be measured accurately with the rat wild-type (WT) olfactory cyclic nucleotide-gated (CNG) channel (See,
As described in greater detail herein, the present invention provides modified CNG channels to increase its sensitivity and specificity for cAMP and to remove regulation by Ca2+-CaM. The present invention further provides convenient optical methods for detecting changes in cAMP, taking advantage of the Ca2+ permeability of the channel. Using the modified CNG channels, this assay is sensitive to cAMP in the physiologic range (0.1-50 μM). As described herein, this approach has been used to probe the interactions between adenylyl cyclase (AC) and PDE in regulating local cAMP concentration in two cell types, nonexcitable HEK-293 and excitable GH4C1 pituitary cells.
cAMP and Signalling
Much has been learned about the enzymes involved in the generation and breakdown of cAMP (Sunahara et al., Ann. Rev. Pharmacol. Toxicol., 36:461-480 [1996]; and Beavo, Physiol. Rev., 75:725-748 [1995]), and the proteins that mediate the downstream effects of cAMP (Walsh et al., J. Biol. Chem., 243:3763-3765 [1968]; Gray et al., Curr. Opin. Neurobiol., 8:330-334 [1998]; Francis and Corbin, Crit. Rev. Clin. Lab. Sci., 36:275-328 [1999]; and Finn et al., Ann. Rev. Physiol., 58:395-426 [1996]). However, conventional methods have, by and large, been unable to resolve the spatial and temporal features of cAMP signals. Creative attempts to use protein kinase A (PKA) or L-type Ca2+ channels to assess changes in cAMP levels have yielded some information. In large invertebrate neurons gradients in cAMP between the processes and the soma have been inferred (Bacskai et al., Science 260:222-226 [1993]; and Hempel et al., Nature 384:166-169 [1996]). Gradients in cAMP have also been deduced in studies of cardiac myocytes, in which only part of the cell was stimulated with isoproterenol (Jurevicius and Fischmeister, Proc. Natl. Acad. Sci. USA 93:295-299 [1996]). Unfortunately, these methods have limited resolution. It has been shown previously that heterologously-expressed, cyclic nucleotide-gated (CNG) channels are able to detect changes in membrane-localized cAMP concentration (Rich et al., J. Gen. Physiol., 116:147-161 [2000]; Rich et al., J. Gen. Physiol., 118:63-77; and Fagan et al., FEBS Lett., 500:85-90 [2001]) and, using the patch-cram technique, cytosolic cGMP (Trivedii and Kramer, Neuron 21:895-906 [1998]) in real time.
In contrast, in some embodiments of the present invention, the properties of CNG channels are tailored for the measurement of submicromolar cAMP concentrations (Rich et al. [2001], supra). These methodological advances allow the measurement of cAMP produced by low levels of adenylyl cyclase (AC) activity without the need to inhibit phosphodiesterase (PDE) (Rich et al. [2001], supra; and Fagan et al., supra).
During the development of the present invention, comparisons of the measurements of membrane-localized and total cellular cAMP levels in response to prostaglandin E1 (PGE1) stimulation were made. The olfactory CNG channel α subunit containing two mutations, C460W and E583M, were used to monitor cAMP levels near the surface membrane in both cell populations and single cells. These mutations make it possible to measure cAMP concentrations in the 100 nM range, while rendering the channel relatively insensitive to cGMP. Total cAMP accumulation in cell populations was investigated by measuring the conversion of [3H]ATP into [3H]cAMP. The difference between the two measured cAMP signals was striking: a rise and fall in cAMP near the membrane and an increase to a steady level throughout the cell. The segregation of signals helps to explain how cAMP can differentially regulate cellular targets.
Indeed, results provided herein indicate that cAMP signals near the channels are distinct from those in other parts of the cell. Several lines of evidence presented previously suggest that cAMP is produced in subcellular compartments near the surface membrane, and that diffusion between these domains and a cytosolic compartment is significantly impeded (Rich et al., [2000], supra). At a minimum, it is necessary to invoke the same two compartments to explain the current data. With no restriction on diffusion: 1) cAMP concentrations right next to AC are not high enough to activate PKA (Rich et al. [2000], supra), let alone the CNG channels used here (unless the entire cell filled with cAMP); and 2) cAMP would diffuse across a 15-20 μm cell in <0.2 s, which would ‘wash out’ any spatial differences on a time-scale of tens to hundreds of seconds. The inset to
where V1 and V2 are the volumes of compartments 1 and 2, C1 and C2 are the cAMP concentrations, J12 is the flux coefficient between compartments, EAC is the synthesis rate of cAMP, E1 and E2 are the maximal cAMP hydrolysis rates, KM1 and KM2 are the Michaelis constants for PDE activity, A and I are the fraction of active and inactive PDE in compartment 1 (A+I=1), and kA and kI, are the rate constants of PDE activation and inactivation. The parameters J12=8.0×10−16 L/s, V1=0.040 pL, and V2=2.0 pL are the same as those used previously (Rich et al., [2000], supra). AC activity is considered constant, with EAC=0.13 μM/s. KM1, E1, KM2, and E2 are 0.30 μM, 0.83 μM/s, 1.0 μM, and 0.0020 μM/s. The rate constants kA and kI, are 0.0015 s−1 and 0.0010 s−1. The initial and final (300 s) values of A are 0.10 and 0.36. The parameters used here reflect similar total PDE activities near the plasma membrane and throughout the cytosol, in broad agreement with experiments on PDE type IV in several cell types (Houslay et al., Adv. Pharmacol., 44:225-342 [1998]).
Simulations of the model successfully reproduce the local transient change in cAMP, as well as the rise in total cAMP to a plateau (See,
In morphologically-complex cells like neurons, dendritic spines and other subcellular structures have been shown to be diffusionally isolated compartments (Svoboda et al., Science 272:716-719 [1996]; Bridge et al., Science 248:376-378 [1990]; Leblanc and Hume, Science 248:372-376 [1990]; Finch and Augustine, Nature 396:753-756 [1998]; and Takechi et al., Nature 396:757-760 [1998]). Surprisingly, results obtained during the development of the present invention indicate that the concept of three-dimensional barriers to diffusion may be generalized to simple cells, which are usually considered to contain a single homogeneous cytosolic compartment. The exact nature of the diffusional barrier is unclear, but it is likely to be formed, at least in part, by ER membrane that is known to come in close apposition to the plasma membrane (Ma et al., Science 287:1647-1651 [2000]; Akita and Kuba, J. Gen. Physiol., 116:697-720 [2000]; and Martin and Fuchs, Proc. Roy. Soc. London B. Biol. Sci., 250:71-76 [1992]). Although localized Ca2+ signals have been measured (Roberts et al., J. Neurosci., 10:3664-3684 [1990]; and Jaggar et al., Am. J. Physiol. Cell Physiol., 278:C235-C256 [2000]), these have been attributed primarily to proximity to high-throughput Ca2+ sources and the delimiting effects of high-capacity cellular buffers, rather than diffusional barriers. However, it is contemplated that the diffusional barriers to cAMP described herein also influence local Ca2+ signals. Regardless, an understanding of the mechanism(s) is not necessary in order to use the present invention.
Thus, experiments conducted during the development of the present invention have resolved distinct cAMP signals in different compartments of a simple, nonexcitable cell: a transient signal in microdomains near the surface membrane and a signal that rises to a plateau throughout the cell. Diffusional restrictions between the microdomains and the cytosol, as well as differential regulation of PDE activity, are required to generate such distinct signals. Segregated cAMP signals allow for differential regulation of cAMP effector proteins like PKA. This may explain a long-standing observation in cardiac myocytes: two agents that both trigger rises in cAMP (PGE1 and isoproterenol) have markedly different downstream effects (Steinberg and Brunton, Ann. Rev. Pharmacol. Toxicol., 41:751-773 [2001]). Similar observations have been made in comparing the effects of β1- and β2-adrenergic agonists (Steinberg and Brunton, Ann. Rev. Pharmacol. Toxicol., 41:751-773 [2001]; Chen-Izu et al., Biophys. J., 79:2547-2556 [2000]; and Davare et al., Science 293:98-101 [2001]). In this context, it is interesting to reconsider the possible functions of A-kinase anchoring proteins (AKAPs), the scaffolds that tether PKA to cellular targets (Feliciello et al., J. Mol. Biol., 308:99-114 [2001]; and Gray et al., Curr. Opin. Neurobiol., 8:330-334 [1998]). In addition to creating two-dimensional protein arrays that help to ensure the phosphorylation of certain proteins, AKAPs are likely to direct PKA to diffusionally isolated cellular regions in which distinct cAMP signals are produced. The transient nature of the signals measured here limits the diffusional spread of cAMP. However, these signals should still allow for a prolonged activation of PKA because the reassociation of PKA subunits is slow (Ogried and Doskeland, Biochem., 22:1686-1696 [1983]; and Harootunian et al., Mol. Biol. Cell 4:993-1002 [1993]).
Improving the cAMP-Sensing Capabilities of CNG Channels
During the development of the present invention, experiments indicated that adenovirus-expressed rat olfactory cyclic CNG channels have several properties that make them excellent cAMP sensors. These properties include their location at the plasma membrane, rapid gating kinetics, and lack of desensitization. Furthermore, these channels appear to co-localize with AC in discrete regions of the membrane, which allows the measurement of localized cAMP signals. However, there are several limitations to the use of WT CNG channels to detect changes in cAMP. First, WT channels have a low apparent affinity for cAMP (Table 1, below and
First, the mutation E583M was introduced into the rat olfactory CNG channel α subunit (See,
To further increase the sensitivity to cAMP, a novel second mutant, C460W/E583M, was constructed (See,
To remove regulation of the channel by Ca2+-CaM binding residues 61 through 90 were deleted using method previously described (Liu et al., Science 266:1348-1354 [1994]). This channel, Δ61-90/C460W/E583M (See,
In sum, the modified CNG channels of the present invention provide the first ion channels that have been tailored for the measurement of cAMP, as they are primarily activated by cAMP. Although other mutations have been previously made at C460, the present invention provides mutations that exhibit greater changes in cAMP sensitivity. Furthermore, mutations at these positions have apparently never been combined. As little is known about the three-dimensional structure of CNG channels, there is no way to predict the effects of the combined mutations. In addition, the deletion of residues 61-90, which results in the removal of Ca2+-CaM sensitivity, dramatically lowers cyclic nucleotide sensitivity (i.e., 10- to 20-fold). Thus, the prior art teaches away from the mutations of the present invention and their use in assay systems with increased sensitivity for cAMP measurements.
Assessment of the Relative cAMP Sensitivity of Channel Constructs In Vivo
Next, experiments were conducted to assess the ability of each channel construct to detect increases in local cAMP concentration. Changes in cAMP concentration were detected using fura-2 to monitor Ca2+ influx through CNG channels, as described in the Examples. Ca2+ influx induced by different concentrations of forskolin, an AC activator, in the presence of 100 μM IBMX, a nonselective PDE inhibitor was measured. This approach allowed comparisons to be made between the cAMP sensitivities of the different channels to changes in cAMP, regardless of variations in expression levels between experiments. In HEK-293 cells expressing the WT channel, addition of forskolin was followed by a brief delay and an increase in Ca2+ influx (See,
PDE Activity in HEK-293 Cells
As discussed herein, CNG channels monitor cAMP produced in subcellular compartments near the plasma membrane. To assess the extent to which PDE activity affects cAMP levels, forskolin-induced Ca2+ influx in the presence and absence of PDE inhibitors was measured. Initially the effects of the nonselective PDE inhibitor, IBMX, were examined using each CNG channel construct as a cAMP sensor. In cells expressing the WT channel, there was little or no change in Ca2+ influx in response to an intermediate forskolin concentration (10 μM; See,
Next, experiments were conducted to pharmacologically identify the PDE type(s) that regulates cAMP levels near CNG channels. The C460W/E583M channels were used to monitor forskolin-stimulated cAMP accumulation in the presence and absence of a series of PDE inhibitors. The reported IC50 values of inhibitors used in this study for each PDE type are given in Table 3. The PDE inhibitor concentrations used were typically at least 5-fold higher than the most potent IC50. Either vehicle or PDE inhibitors were added at 0 s and 1 μM forskolin was added at 180 s (See,
In Vivo Estimates of PDE Inhibitor KI
To further establish that PDE type IV is responsible for the observed IBMX-sensitive PDE activity, the KI's (inhibition constants) of IBMX and two PDE type IV inhibitors (RO-20-1724 and rolipram) were estimated in vivo. This required developing a quantitative framework to assess the relationship between cAMP synthesis, hydrolysis, and redistribution throughout the cell. Thus, the following formalism was adopted:
where C is the steady-state rate of cAMP synthesis by AC, Vmax is the maximal rate of cAMP hydrolysis, Km is the Michaelis constant for PDE, and kf is the rate constant of cAMP flux out of the microdomain. In order to estimate KI for the PDE inhibitors two assumptions were made. First, it was assumed that at low levels of AC stimulation and PDE inhibition, the concentration of local cAMP is low and diffusion of cAMP out of the microdomain is negligible. Second, the assumption was made that cAMP levels reach steady-state shortly after AC stimulation (i.e., equal rates of synthesis and hydrolysis). With these assumptions, Eq. 4 can be simplified to:
Interestingly, this equation reveals that when the inhibitor concentration is equal to KI, the cAMP concentration is twice that in the absence of inhibitor. At low cAMP concentrations (<K1/2 for the channel), the cAMP concentration is proportional to the square root of the Ca2+ influx rate (the Hill coefficient for channel activation is approximately 2). Thus, KI, can be estimated using a linear fit to the square root of the slopes of the fluorescence traces as a function of inhibitor concentration (See,
The high cAMP affinity C460W/E583M channels were used to detect changes in cAMP concentration following pretreatment with PDE inhibitors (added at 0 s) and modest forskolin stimulation (0.5 μM added at 180 s). IBMX, RO-20-1724, and rolipram were completely equilibrated across the plasma membrane of HEK-293 cells in <180 s (data not shown). The assumption that cAMP levels reach steady-state is supported by the long-lasting linear rise in Ca2+ concentration following forskolin stimulation (a steady Ca2+ influx rate reflects a constant cAMP level; See,
At higher PDE inhibitor concentrations, the square root of Ca2+ influx rate, which is proportional to cAMP concentration, deviates from linearity (See,
PDE Activity in GH4C1 Cells
To further test the utility of this approach, PDE activity in excitable GH4C1 pituitary cells was examined. In all of the experiments shown (See,
Next, forskolin-induced responses and the effects of IBMX in cells expressing the high cAMP affinity construct, C460W/E583M were examined. In these experiments either vehicle or 100 μM IBMX was added at 60 s (
To identify the PDE family or families that regulate the local cAMP concentration in GH4C1 cells, cAMP accumulation was monitored in the presence and absence of PDE inhibitors (See, Table 2). Again, the high cAMP affinity C460W/E583M channels were used. PDE inhibitors were added at 60 s. Although the addition of IBMX alone triggered Ca2+ influx, 10 μM forskolin were added at 240 s as a positive control for CNG channel activity. Only the nonspecific PDE inhibitor, IBMX, and the PDE-type-I specific inhibitor, 8-methoxymethyl-IBMX, induced Ca2+ influx through CNG channels (Table 2). This PDE is likely to have a low Km for cAMP because it is capable of regulating cAMP at concentrations too low for the WT or Δ61-90/C460W/E583M channels to detect. Thus, it is contemplated that a Ca2+-CaM stimulated PDE (type I) is primarily responsible for controlling basal cAMP signals in GH4C1 cells.
In addition, PDE activity was monitored using Δ61-90/C460W/E583M channels to detect changes in cAMP concentration (See,
The methods described herein are suitable for determination of whether an unknown compound modulates PDE activity or adenylyl cyclase activity. Known inhibitors of PDE (either type-specific inhibitors described above or non-type-specific inhibitors such as IBMX) are mixed, at saturating concentrations, with the unknown compound. If the known PDE inhibitors (particularly type-specific ones) block the effect of the unknown compound (e.g., a rise in cAMP detected as described herein), then the unknown compound is categorized as a candidate PDE modulator. If a series of PDE inhibitors do not prevent the effect of the unknown compound, then the compound is categorized as a candidate adenylyl cyclase modulator. These initial determinations are then verified by using more difficult and time-consuming assays with purified proteins. Thus, the methods described herein provide very useful initial screening tools for compounds that are capable of modulating PDE or adenylyl cyclase activity in living cells.
Definitions
As used herein, the terms “patch clamp” and “patch clamp recording methods” refer to methods that involve sealing the tip of a small (e.g., about 1 micron) orifice glass pipette to the membrane of a cell. Under optimal conditions, a seal resistance of greater than one billion ohms is formed around the rim of the pipette tip between the cell membrane and the glass. When the pipette is connected to an appropriate amplifier, small currents across the patch of membrane inside the pipette tip can be recorded. This initial configuration is referred to as a “cell-attached patch.” When such a patch is pulled from the cell, an “excised inside-out patch” forms, with the cytoplasmic face of the patch membrane facing the bathing solution. Alternatively, in the cell-attached configuration, the membrane inside the patch pipette can be ruptured with light suction to provide access to the cell cytoplasm. In this condition, currents are recorded from the entire cell (i.e., “whole-cell configuration”). In the “perforated patch configuration,” a pore-forming antimicrobial (e.g., nystatin) is added to the pipette solution to gain electrical access to the cell's interior. This allows currents to be recorded from the entire cell, while retaining divalent cations and larger molecules (e.g., cAMP) in the cell.
As used herein, the term “adenoviruses” (Ad) refers to the double-stranded DNA viruses of the Adenoviridae. The genome of adenoviruses (˜36 kb) is complex and contains over 50 open reading frames (ORFs). These ORFs are overlapping and genes encoding one protein are often embedded within genes coding for other Ad proteins. Expression of Ad genes is divided into an early and a late phase. Early genes are those transcribed prior to replication of the genome while late genes are transcribed after replication. The early genes comprise E1a, E1b, E2a, E2b, E3 and E4. The E1a gene products are involved in transcriptional regulation, while the E1b gene products are involved in the shut-off of host cell functions and mRNA transport. E2a encodes the a DNA-binding protein (DBP), while E2b encodes the viral DNA polymerase and preterminal protein (pTP). The E3 gene products are not essential for viral growth in cell culture. The E4 region encodes regulatory protein involved in transcriptional and post-transcriptional regulation of viral gene expression, and subset of the E4 proteins are essential for viral growth. The products of the late genes (e.g., L1-5) are predominantly components of the virion as well as proteins involved in the assembly of virions. The VA genes produce VA RNAs which block the host cell from shutting down viral protein synthesis.
Adenoviruses or recombinant Ad vectors have been exploited for the delivery of foreign genes to cells for a number of reasons including the fact that Ad vectors have been shown to be highly effective for the transfer of genes into a wide variety of tissues in vivo and the fact that Ad infects both dividing and non-dividing cells.
As used herein, the term “virus” refers to obligate, ultramicroscopic, intracellular parasites incapable of autonomous replication (i.e., replication requires the use of the host cell's machinery). Adenoviruses, as noted above, are double-stranded DNA viruses. The left and right inverted terminal repeats (ITRs) are short elements located at the 5′ and 3′ termini of the linear Ad genome, respectively and are required for replication of the viral DNA. The two ITRs are inverted repeats of each other. The “adenovirus packaging sequence” refers to the sequence which comprises five (AI-AV) packaging signals and is required for encapsidation of the mature linear genome; the packaging signals are located from ˜194 to 358 bp in the Ad genome (about 0.5-1.0 mu).
As used herein, the term “primary cell” refers to a cell which is directly obtained from a tissue or organ of an animal whether or not the cell is in culture.
As used herein, the term “cultured cell” refers a cell which has been maintained and/or propagated in vitro. Cultured cells include primary cultured cells and cell lines.
As used herein, the term “primary cultured cells” refers to primary cells which are cultured in vitro and which preferably, though not necessarily, are capable of undergoing ten or fewer passages in in vitro culture before senescence and/or cessation of proliferation.
As used herein, the terms “cell line” and “immortalized cell” refer to a cell which is capable of a greater number of cell divisions in vitro before cessation of proliferation and/or senescence as compared to a primary cell from the same source. A cell line includes, but does not require, that the cells be capable of an infinite number of cell divisions in culture. The number of cell divisions may be determined by the number of times a cell population may be passaged (i.e., subcultured) in in vitro culture. Passaging of cells is accomplished by methods known in the art. Cell lines may be generated spontaneously or by transformation. A “spontaneous cell line” is a cell line which arises during routine culture of cells. A “transformed cell line” refers to a cell line which is generated by the introduction of a “transgene” comprising nucleic acid (usually DNA) into a primary cell or into a finite cell line by means of human intervention
Cell lines include, but are not limited to, finite cell lines and continuous cell lines. As used herein, the term “finite cell line” refers to a cell line which is capable of a limited number (from about 1 to about 50, more preferably from about 1 to about 40, and most preferably from about 1 to about 20) of cell divisions prior to senescence. The term “continuous cell line” refer to a cell line which is capable of more than about 50 (and more preferably, an infinite number of) cell divisions. A continuous cell line generally, although not necessarily, also has the general characteristics of a reduced cell size, higher growth rate, higher cloning efficiency, increased tumorigenicity, and/or a variable chromosomal complement as compared to the finite cell line or primary cultured cells from which it is derived.
The term “transgene” as used herein refers to any nucleic acid sequence which is introduced into the cell by experimental manipulations. A transgene may be an “endogenous DNA sequence” or a “heterologous DNA sequence” (i.e., “foreign DNA”). The term “endogenous DNA sequence” refers to a nucleotide sequence which is naturally found in the cell into which it is introduced so long as it does not contain some modification (e.g., a point mutation, the presence of a selectable marker gene, etc.) relative to the naturally-occurring sequence. The term “heterologous DNA sequence” refers to a nucleotide sequence which is ligated to, or is manipulated to become ligated to, a nucleic acid sequence to which it is not ligated in nature, or to which it is ligated at a different location in nature. Heterologous DNA is not endogenous to the cell into which it is introduced, but has been obtained from another cell. Heterologous DNA also includes an endogenous DNA sequence which contains some modification. Generally, although not necessarily, heterologous DNA encodes RNA and proteins that are not normally produced by the cell into which it is expressed. Examples of heterologous DNA include reporter genes, transcriptional and translational regulatory sequences, selectable marker proteins (e.g., proteins which confer drug resistance), etc.
The term “label” as used herein refers to any atom or molecule which can be used to provide a detectable (preferably quantifiable) signal, and which can be attached to a nucleic acid or protein. Labels may provide signals detectable by fluorescence, radioactivity, colorimetry, gravimetry, X-ray diffraction or absorption, magnetism, enzymatic activity, and the like.
As used herein, the term “detection signal” refers to an indicator of the presence or absence of a compound of interest. For example, in some methods of the present invention, fluorescence (i.e., the detection signal) is measured at particular excitation and emission wavelengths, to detect Ca2+ influx. In these methods, Ca2+ influx was found to cause a decrease in fluorescence (ΔF), which was expressed relative to the pre-stimulus fluorescence (F0) to correct for variations in dye concentration, and to allow for comparison of results on different batches of cells. Although fluorescence was used in the Examples described herein, it is not intended that the present invention be limited to systems that rely upon use of a fluorescent signal. Indeed, assay systems utilizing other signals find use in the present invention, including but not limited to chemiluminescence, bioluminescence, etc., as known in the art.
As used herein, the term “second messenger” refers to small molecules or ions that are generated in the cytoplasm of cells in response to binding of a signal molecule to its receptor on the outer surface of the cell membrane. Two major classes of second messengers are known, including one in which cAMP is involved and one which involves a combination of calcium ions and either inositol triphosphate or diacylglycerol.
As used herein, the term “ion channel” refers to a pathway (i.e., a channel) through the cell membrane, which allows ions to enter and/or exit cells.
As used herein, the term “gated ion channel” refers to ion channels that exhibit selectivity in the timing and/or properties of ions that are allowed to pass through an ion channel and enter or exit a cell. Typically, these channels have a structure which determines the particles that are allowed to enter and/or exit the cell.
The terms “functional calcium channel” and “biologically active calcium channel” interchangeably refer to a calcium channel which allows entry into a cell of a calcium ion in response to a stimulus. Such entry may be determined by measuring the amount of current which flows through the calcium channel in response to the stimulus. Alternatively, a functional calcium channel refers to a calcium channel which binds ligands that have affinity for a calcium channel. For ligand binding assays of a recombinant calcium channel, it is preferred that the host cell which is used for testing the function of the recombinant calcium channel not produce endogenous calcium channel subunits that are of a type or in an amount that interferes with the detection of the recombinant calcium channel. Methods for determining the function of a calcium channel are known in the art.
The term “compound that modulates calcium channel activity” and grammatical equivalents thereof refers to a compound that alters (i.e., reduces or increases) the ability of a calcium channel to pass calcium ions as measured by, for example, the current flowing through the calcium channel. Such compounds include, but are not limited to, calcium channel agonists (e.g., Goldin et al., U.S. Pat. No. 5,312,928, herein incorporated by reference) and antagonists, and compounds that exert their effect on the activity of the calcium channel directly or indirectly.
As used herein, the term “permeant” refers to molecules which are capable of entering cells by means of ion channels or other mechanisms. “Permeant” includes, but is not limited to ions such as chloride, potassium, sodium, and thiocyanate.
The term “biologically active” as used herein, refers to a protein or other biologically active molecules (e.g., catalytic RNA) having structural, regulatory, or biochemical functions of a naturally occurring molecule. Likewise, immunologically active refers to the capability of the natural, recombinant, or synthetic human protein, or any oligopeptide or polynucleotide thereof, to induce a specific immune response in appropriate animals or cells and to bind with specific antibodies.
As used herein, the term “agonist” refers to molecules or compounds which mimic the action of a “native” or “natural” compound. Agonists may be homologous to these natural compounds in respect to conformation, charge or other characteristics. Thus, agonists may be recognized by receptors expressed on cell surfaces. This recognition may result in physiologic and/or biochemical changes within the cell, such that the cell reacts to the presence of the agonist in the same manner as if the natural compound was present. Agonists may include proteins, nucleic acids, carbohydrates, or any other molecules which bind or interact with gated ion channels.
As used herein, the terms “antagonist” and “inhibitor” refer to molecules or compounds which inhibit the action of a “native” or “natural” compound. Antagonists may or may not be homologous to these natural compounds in respect to conformation, charge or other characteristics. Thus, antagonists may be recognized by the same or different receptors that are recognized by an agonist. Antagonists may have allosteric effects which prevent the action of an agonist (e.g., prevent opening of the chloride ion channel). Or, antagonists may prevent the function of the agonist (e.g., by blocking the passage of chloride ions in the channels). In contrast to the agonists, antagonistic compounds do not result in physiologic and/or biochemical changes within the cell such that the cell reacts to the presence of the antagonist in the same manner as if the natural compound was present. Antagonists and inhibitors may include proteins, nucleic acids, carbohydrates, or any other molecules which bind or interact with gated ion channels. As used herein, the term modulate, refers to a change or an alteration in the biological activity. Modulation may be an increase or a decrease in protein activity, a change in binding characteristics, a change in ion passage through a gated channel, or any other change in the biological, functional, or immunological properties associated with the activity of a protein or other structure of interest.
As used herein, the term “transformation” refers to the introduction of foreign genetic material into a cell or organism. Transformation may be accomplished by any method known in the art which permits the successful introduction of nucleic acids into cells and which results in the expression of the introduced nucleic acid. For example, transformation may be used to introduce cloned DNA encoding a normal or mutant cyclic nucleotide gated ion channel into a cell which normally does not express this ion channel. Transformation may be accomplished through use of any expression vector. For example, the use of baculovirus to introduce foreign nucleic acid into insect cells is contemplated. The term “transformation” also includes methods such as P-element mediated germline transformation of whole insects. As used herein, the term “transformation” also includes but is not limited to methods such as “transfection” and “transduction.”
The term “transfection” generally refers to the introduction of foreign DNA into eukaryotic cells, but may also be used to refer to the introduction of foreign DNA into prokaryotic cells. Transfection may be accomplished by a variety of means known to the art including but not limited to calcium phosphate-DNA co-precipitation, DEAE-dextran-mediated transfection, polybrene-mediated transfection, electroporation, microinjection, liposome fusion, lipofection, protoplast fusion, viral infection (transduction), and biolistics.
As used herein, the term “gene” refers to the deoxyribonucleotide sequences comprising the coding region of a structural gene and including sequences located adjacent to the coding region on both the 5′ and 3′ ends for a distance of several kb on either end such that the gene corresponds to the length of the full-length mRNA. The sequences which are located 5′ of the coding region and which are present on the mRNA are referred to as 5′ non-translated sequences. The sequences which are located 3′ or downstream of the coding region and which are present on the mRNA are referred to as 3′ non-translated sequences. A genomic form or clone of a gene contains coding sequences, termed exons, alternating with non-coding sequences termed “introns” or “intervening regions” or “intervening sequences.” Introns are segments of a gene which are transcribed into heterogenous nuclear RNA (mRNA); introns may contain regulatory elements such as enhancers. Introns are removed or “spliced out” from the nuclear or primary transcript; introns therefore are absent in the messenger RNA (mRNA) transcript. In addition to containing introns, genomic forms of a gene may also include sequences located on both the 5′ and 3′ end of the sequences which are present on the RNA transcript. These sequences are referred to as “flanking” sequences or regions (these flanking sequences are located 5′ or 3′ to the non-translated sequences present on the mRNA transcript). The 5′ flanking region may contain regulatory sequences such as promoters and enhancers which control or influence the transcription of the gene. The 3′ flanking region may contain sequences which direct the termination of transcription, posttranscriptional cleavage and polyadenylation.
As used herein, the term “coding region,” when used in reference to a structural gene refers to the nucleotide sequences which encode the amino acids found in the nascent polypeptide as a result of translation of an mRNA molecule. The coding region is bounded, in eukaryotes, on the 5′ side by the nucleotide triplet “ATG” which encodes the initiator methionine and on the 3′ side by one of the three triplets which specify stop codons (i.e., TAA, TAG, TGA).
As used herein, the term “structural gene” refers to a DNA sequence coding for RNA or a protein. In contrast, “regulatory genes” are structural genes which encode products (e.g., transcription factors) which control the expression of other genes.
As used herein, the term “regulatory element” refers to a genetic element which controls some aspect of the expression of nucleic acid sequences. For example, a promoter is a regulatory element which facilitates the initiation of transcription of an operably linked coding region. Other regulatory elements are splicing signals, polyadenylation signals, termination signals, enhancer elements, etc. Promoters and enhancers consist of short arrays of DNA sequences that interact specifically with cellular proteins involved in transcription (Maniatis et al., Science 236:1237 [1987]). Promoter and enhancer elements have been isolated from a variety of eukaryotic sources including genes in yeast, insect and mammalian cells and viruses (analogous control elements [i.e., promoters], are also found in prokaryotes). The selection of a particular promoter and enhancer depends on what cell type is to be used to express the protein of interest. Some eukaryotic promoters and enhancers have a broad host range while others are functional in a limited subset of cell types (for review see Voss, et al., Trends Biochem. Sci., 11:287 [1986]; and Maniatis, et al., Science 236:1237 [1987]). For example, the SV40 early gene enhancer is very active in a wide variety of cell types from many mammalian species and has been widely used for the expression of proteins in mammalian cells (Dijkema, et al., EMBO J. 4:761 [1985]). Other examples of promoter/enhancer elements active in a broad range of mammalian cell types are those from the human elongation factor 1 gene (Uetsuki et al., J. Biol. Chem., 264:5791 [1989]; Kim et al., Gene 91:217 [1990]; and Mizushima and Nagata, Nucl. Acids. Res., 18:5322 ([990]) and the long terminal repeats of the Rous sarcoma virus (Gorman et al., Proc. Natl. Acad. Sci. USA 79:6777 [1982]) and the human cytomegalovirus (Boshart et al., Cell 41:521 [1985]).
The terms “promoter element” and “promoter” as used herein refer to a DNA sequence that is located at the 5′ end of (i.e., precedes) a gene in a DNA polymer and provides a site for initiation of the transcription of the gene into mRNA.
The terms “gene of interest” and “nucleotide sequence of interest” refer to any gene or nucleotide sequence, respectively, the manipulation of which may be deemed desirable for any reason by one of ordinary skill in the art.
The term “expression vector” as used herein refers to a recombinant DNA molecule containing a desired coding sequence and appropriate nucleic acid sequences necessary for the expression of the operably linked coding sequence in a particular host organism. Nucleic acid sequences necessary for expression in prokaryotes include a promoter, optionally an operator sequence, a ribosome binding site and possibly other sequences. Eukaryotic cells are known to utilize promoters, enhancers, and termination and polyadenylation signals.
A “modification” as used herein in reference to a nucleic acid sequence refers to any change in the structure of the nucleic acid sequence. Changes in the structure of a nucleic acid sequence include changes in the covalent and non-covalent bonds in the nucleic acid sequence. Illustrative of these changes are mutations, mismatches, strand breaks, as well as covalent and non-covalent interactions between a nucleic acid sequence (which contains unmodified and/or modified nucleic acids) and other molecules. Illustrative of a covalent interaction between a nucleic acid sequence and another molecule are changes to a nucleotide base (e.g., formation of thymine glycol) and covalent cross-links between double-stranded DNA sequences which are introduced by, for example, ultraviolet radiation or by cis-platinum. Yet another example of a covalent interaction between a nucleic acid sequence and another molecule includes covalent binding of two nucleic acid sequences to psoralen following ultraviolet irradiation. Non-covalent interactions between a nucleic acid sequence and another molecule include non-covalent interactions of a nucleic acid sequence with a molecule other than a nucleic acid sequence and other than a polypeptide sequence. Non-covalent interactions between a nucleic acid sequence with a molecule other than a nucleic acid sequence and other than a polypeptide sequence are illustrated by non-covalent intercalation of ethidium bromide or of psoralen between the two strands of a double-stranded deoxyribonucleic acid sequence. The present invention contemplates modifications which cause changes in a functional property (or properties), such changes manifesting themselves at a variety of time points.
The term “allelic series” when made in reference to a gene refers to wild-type sequences of the gene. An “allelic series of modifications” as used herein in reference to a gene refers to two or more nucleic acid sequences of the gene, where each of the two or more nucleic acid sequences of the gene contains at least one modification when compared to the wild-type sequences of the gene.
As used herein, the term “mutation” refers to a deletion, insertion, or substitution. A “deletion” is defined as a change in a nucleic acid sequence in which one or more nucleotides is absent. An “insertion” or “addition” is that change in a nucleic acid sequence which has resulted in the addition of one or more nucleotides. A “substitution” results from the replacement of one or more nucleotides by a molecule which is a different molecule from the replaced one or more nucleotides. For example, a nucleic acid may be replaced by a different nucleic acid as exemplified by replacement of a thymine by a cytosine, adenine, guanine, or uridine. Alternatively, a nucleic acid may be replaced by a modified nucleic acid as exemplified by replacement of a thymine by thymine glycol.
The term “mismatch” refers to a non-covalent interaction between two nucleic acids, each nucleic acid residing on a different polynucleic acid sequence, which does not follow the base-pairing rules. For example, for the partially complementary sequences 5′-AGT-3′ and 5′-AAT-3′, a G-A mismatch is present.
The terms “nucleic acid” and “unmodified nucleic acid” as used herein refer to any one of the known four deoxyribonucleic acid bases (i.e., guanine, adenine, cytosine, and thymine). The term “modified nucleic acid” refers to a nucleic acid whose structure is altered relative to the structure of the unmodified nucleic acid. Illustrative of such modifications would be replacement covalent modifications of the bases, such as alkylation of amino and ring nitrogens as well as saturation of double bonds.
The term “modified cell” refers to a cell which contains at least one modification in the cell's genomic sequence.
The term “nucleic acid sequence-modifying agent” refers to an agent which is capable of introducing at least one modification into a nucleic acid sequence. Nucleic acid sequence-modifying agents include, but are not limited to, chemical compounds (e.g., N-ethyl-N-nitrosurea (ENU), methylnitrosourea (MNU), procarbazine hydrochloride (PRC), triethylene melamine (TEM), acrylamide monomer (AA), chlorambucil (CHL), melphalan (MLP), cyclophosphamide (CPP), diethyl sulfate (DES), ethyl methane sulfonate (EMS), methyl methane sulfonate (MMS), 6-mercaptopurine (6 MP), mitomycin-C (MMC), procarbazine (PRC), N-methyl-N′-nitro-N-nitrosoguanidine (MNNG), 3H2O, and urethane (UR)), and electromagnetic radiation (e.g., X-ray radiation, gamma-radiation, ultraviolet light).
The term “wild-type” when made in reference to a gene refers to a gene which has the characteristics of that gene when isolated from a naturally occurring source. A wild-type gene is that which is most frequently observed in a population and is thus arbitrarily designated the “normal” or “wild-type” form of the gene. In contrast, the term “modified” or “mutant” refers to a gene or gene product which displays modifications in sequence and/or functional properties (i.e., altered characteristics) when compared to the wild-type gene or gene product. It is noted that naturally-occurring mutants can be isolated; these are identified by the fact that they have altered characteristics when compared to the wild-type gene or gene product.
A “variant of CNG” is defined as an amino acid sequence which differs by one or more amino acids from the wild-type CNG sequence. The variant may have conservative changes, wherein a substituted amino acid has similar structural or chemical properties (e.g., replacement of leucine with isoleucine). More rarely, a variant may have nonconservative changes (e.g., replacement of a glycine with a tryptophan). Similar minor variations may also include amino acid deletions or insertions (i.e., additions), or both. A variant CNG can be engineered using any number of molecular biology techniques known in the art including but not limited tosite-directed mutagenesis of a CNG clone. Variant CNG channels suitable for use with the methods and compositions of the present invention include but are not limited to homologues of the rat olfactory CNG channel (e.g., CNG channels of other species such as mouse and man).
The term “conservative substitution” as used herein refers to a change that takes place within a family of amino acids that are related in their side chains. Genetically encoded amino acids can be divided into four families: (1) acidic (aspartate, glutamate); (2) basic (lysine, arginine, histidine); (3) nonpolar (alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan); and (4) uncharged polar (glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine). Phenylalanine, tryptophan, and tyrosine are sometimes classified jointly as aromatic amino acids. In similar fashion, the amino acid repertoire can be grouped as (1) acidic (aspartate, glutamate); (2) basic (lysine, arginine, histidine), (3) aliphatic (glycine, alanine, valine, leucine, isoleucine, serine, threonine), with serine and threonine optionally be grouped separately as aliphatic-hydroxyl; (4) aromatic (phenylalanine, tyrosine, tryptophan); (5) amide (asparagine, glutamine); and (6) sulfur -containing (cysteine and methionine) (e.g., Stryer ed., Biochemistry, pg. 17-21, 2nd ed, WH Freeman and Co. [1981]). Whether a change in the amino acid sequence of a peptide results in a functional homolog can be readily determined by assessing the ability of the variant peptide to function in a fashion similar to the wild-type protein. Peptides having more than one replacement can readily be tested in the same manner. In contrast, the term “nonconservative substitution” refers to a change in which an amino acid from one family is replaced with an amino acid from another family (e.g., replacement of a glycine with a tryptophan). Guidance in determining which amino acid residues can be substituted, inserted, or deleted without abolishing biological activity can be found using computer programs (e.g., LASERGENE software, DNASTAR Inc., Madison, Wis.).
The terms “targeting vector” and “targeting construct” are used interchangeably to refer to oligonucleotide sequences comprising a gene encoding a cyclic nucleotide gated ion channel. It is preferred that the targeting vector also comprise a selectable marker gene. The targeting vector contains gene sequences sufficient to permit the homologous recombination of the targeting vector into at least one allele of the gene resident in the chromosomes of the target or recipient cell (e.g., ES) cells. Typically, though not necessarily, the targeting vector contains 2 kb to 10 kb of DNA homologous to the gene. This 2 kb to 10 kb of DNA may be located downstream or upstream of the selectable marker gene, or may be divided on each side of the selectable marker gene. In a preferred embodiment, the selectable marker gene is located upstream of the gene. The targeting vector may contain more than one selectable maker gene. When more than one selectable marker gene is employed, the targeting vector preferably contains a positive selectable marker (e.g., the neo gene) and a negative selectable marker (e.g., the diphtheria toxin (dt gene) or Herpes simplex virus tk (HSV-tk) gene). The presence of the positive selectable marker permits the selection of recipient cells containing an integrated copy of the targeting vector whether this integration occurred at the target site or at a random site. The presence of the negative selectable marker permits the identification of recipient cells containing the targeting vector at the targeted site (i.e., which has integrated by virtue of homologous recombination into the target site); cells which survive when grown in medium which selects against the expression of the negative selectable marker do not contain a copy of the negative selectable marker.
In some embodiments, the targeting vectors of the present invention are of the “replacement-type;” integration of a replacement-type vector results in the insertion of a selectable marker into the target gene. Replacement-type targeting vectors may be employed to disrupt a gene resulting in the generation of a null allele (i.e., an allele incapable of expressing a functional protein; null alleles may be generated by deleting a portion of the coding region, deleting the entire gene, introducing an insertion and/or a frameshift mutation, etc.) or may be used to introduce a modification (e.g., one or more point mutations) into a gene.
The terms “selectable marker” or “selectable gene product” as used herein refer to the use of a gene which encodes an enzymatic activity that confers resistance to an antibiotic or drug upon the cell in which the selectable marker is expressed. Selectable markers may be “positive”; positive selectable markers typically are dominant selectable markers (i.e., genes which encode an enzymatic activity which can be detected in any mammalian cell or cell line [including ES cells]). Examples of dominant selectable markers include, but are not limited to, (1) the bacterial aminoglycoside 3′ phosphotransferase gene (also referred to as the neo gene) which confers resistance to the drug G418 in mammalian cells, (2) the bacterial hygromycin G phosphotransferase (hyg) gene which confers resistance to the antibiotic hygromycin and (3) the bacterial xanthine-guanine phosphoribosyl transferase gene (also referred to as the gpt gene) which confers the ability to grow in the presence of mycophenolic acid. Selectable markers may be “negative”; negative selectable markers encode an enzymatic activity whose expression is cytotoxic to the cell when grown in an appropriate selective medium. For example, the HSV-tk gene and the dt gene are commonly used as a negative selectable marker. Expression of the HSV-tk gene in cells grown in the presence of gancyclovir or acyclovir is cytotoxic; thus, growth of cells in selective medium containing gancyclovir or acyclovir selects against cells capable of expressing a functional HSV TK enzyme. Similarly, the expression of the dt gene selects against cells capable of expressing the diphtheria toxin;
An animal whose genome “comprises a heterologous selectable marker gene” is an animal whose genome contains a selectable marker gene not naturally found in the animal's genome which is introduced by means of molecular biological methods. A heterologous selectable marker is distinguished from an endogenous gene naturally found in the animal's genome in that expression or activity of the heterologous selectable marker can be selected for or against.
The terms “in operable combination,” “in operable order,” and “operably linked” as used herein refer to the linkage of nucleic acid sequences in such a manner that a nucleic acid molecule capable of directing the transcription of a given gene and/or the synthesis of a desired protein molecule is produced. The term also refers to the linkage of amino acid sequences in such a manner so that a functional protein is produced.
As used herein, the term “an oligonucleotide having a nucleotide sequence encoding a gene” means a nucleic acid sequence comprising the coding region of a gene (i.e. the nucleic acid sequence which encodes a gene product). The coding region may be present in either a cDNA, genomic DNA or RNA form. When present in a DNA form, the oligonucleotide may be single-stranded (i.e., the sense strand) or double-stranded. Suitable control elements (e.g., enhancers, promoters, splice junctions, polyadenylation signals, etc.) may be placed in close proximity to the coding region of the gene, if needed to permit proper initiation of transcription and/or correct processing of the primary RNA transcript. Alternatively, the coding region utilized in the expression vectors of the present invention may contain endogenous enhancers, splice junctions, intervening sequences, polyadenylation signals, or other sequences, or a combination of both endogenous and exogenous control elements.
The term “an oligonucleotide sequence comprising at least a portion of a cyclic nucleotide gated ion channel gene” refers to a polynucleotide sequence (i.e., a nucleic acid sequence) containing a nucleotide sequence derived from the cyclic nucleotide gated ion channel gene. This sequence may encode a portion of the cyclic nucleotide gated ion channel gene (i.e., not the entire sequence); alternatively, this sequence may encode the entire sequence or may simply contain non-coding regions derived from the gene or a combination of coding and non-coding regions. The oligonucleotide may be RNA or DNA and may be of genomic or synthetic origin.
As used herein the term “portion” when in reference to a gene refers to fragments of that gene. The fragments may range in size from 10 nucleotides to the entire gene sequence minus one nucleotide. Thus, “an oligonucleotide comprising at least a portion of a gene” may comprise small fragments of the gene or nearly the entire gene.
As used herein, the term “substantially purified” refers to molecules, either nucleic or amino acid sequences, that are removed from their natural environment, isolated or separated, and are at least 60% free, preferably 75% free, and most preferably 90% free from other components with which they are naturally associated. An “isolated polynucleotide” is therefore a substantially purified polynucleotide.
As used herein, the term “polymerase chain reaction” (“PCR”) refers to the methods described in U.S. Pat. Nos. 4,683,195, 4,683,202 and 4,965,188, hereby incorporated by reference, which describe methods for increasing the concentration of a segment of a target sequence in a mixture of genomic DNA without cloning or purification. This process for amplifying the target sequence consists of introducing a large excess of two oligonucleotide primers to the DNA mixture containing the desired target sequence, followed by a precise sequence of thermal cycling in the presence of a DNA polymerase. The two primers are complementary to their respective strands of the double stranded target sequence. To effect amplification, the mixture is denatured and the primers then annealed to their complementary sequences within the target molecule. Following annealing, the primers are extended with a polymerase so as to form a new pair of complementary strands. The steps of denaturation, primer annealing and polymerase extension can be repeated many times (i.e., denaturation, annealing and extension constitute one “cycle”; there can be numerous “cycles”) to obtain a high concentration of an amplified segment of the desired target sequence. The length of the amplified segment of the desired target sequence is determined by the relative positions of the primers with respect to each other, and therefore, this length is a controllable parameter. By virtue of the repeating aspect of the process, the method is referred to as the “polymerase chain reaction” (hereinafter “PCR”). Because the desired amplified segments of the target sequence become the predominant sequences (in terms of concentration) in the mixture, they are said to be “PCR amplified”.
With PCR, it is possible to amplify a single copy of a specific target sequence in genomic DNA to a level detectable by several different methodologies (e.g., hybridization with a labeled probe; incorporation of biotinylated primers followed by avidin-enzyme conjugate detection; incorporation of 32P-labeled deoxynucleotide triphosphates, such as dCTP or dATP, into the amplified segment). In addition to genomic DNA, any oligonucleotide sequence can be amplified with the appropriate set of primer molecules. In particular, the amplified segments created by the PCR process itself are, themselves, efficient templates for subsequent PCR amplifications. Amplified target sequences may be used to obtain segments of DNA (e.g., genes) for the construction of targeting vectors, transgenes, etc.
As used herein, the terms “PCR product” and “amplification product” refer to the resultant mixture of compounds after two or more cycles of the PCR steps of denaturation, annealing and extension are complete. These terms encompass the case where there has been amplification of one or more segments of one or more target sequences.
The terms “reverse transcription polymerase chain reaction” and “RT-PCR” refer to a method for reverse transcription of an RNA sequence to generate a mixture of cDNA sequences, followed by increasing the concentration of a desired segment of the transcribed cDNA sequences in the mixture without cloning or purification. Typically, RNA is reverse transcribed using a single primer (e.g., an oligo-dT primer) prior to PCR amplification of the desired segment of the transcribed DNA using two primers.
As used herein, the term “primer” refers to an oligonucleotide, whether occurring naturally as in a purified restriction digest or produced synthetically, which is capable of acting as a point of initiation of synthesis when placed under conditions in which synthesis of a primer extension product which is complementary to a nucleic acid strand is induced, (i.e., in the presence of nucleotides and of an inducing agent such as DNA polymerase and at a suitable temperature and pH). The primer is preferably single stranded for maximum efficiency in amplification, but may alternatively be double stranded. If double stranded, the primer is first treated to separate its strands before being used to prepare extension products. Preferably, the primer is an oligodeoxyribonucleotide. The primer must be sufficiently long to prime the synthesis of extension products in the presence of the inducing agent. The exact lengths of the primers will depend on many factors, including temperature, source of primer and the use of the method.
As used herein, the term “probe” refers to an oligonucleotide (i.e., a sequence of nucleotides), whether occurring naturally as in a purified restriction digest or produced synthetically, recombinantly or by PCR amplification, which is capable of hybridizing to another oligonucleotide of interest. A probe may be single-stranded or double-stranded. Probes are useful in the detection, identification and isolation of particular gene sequences. It is contemplated that any probe used in the present invention will be labeled with any “reporter molecule,” so that it is detectable in any detection system, including, but not limited to enzyme (e.g., ELISA, as well as enzyme-based histochemical assays), fluorescent, radioactive, and luminescent systems. It is not intended that the present invention be limited to any particular detection system or label.
As used herein, the terms “restriction endonucleases” and “restriction enzymes” refer to bacterial enzymes, each of which cut double- or single-stranded nucleic acid at or near a specific nucleotide sequence.
The terms “compound” and “drug candidate” refers to any chemical or biological entity (e.g., including pharmaceuticals, drugs, and the like) that can be used to treat or prevent a disease, illness, sickness, or disorder of bodily function. Compounds comprise both known and potential therapeutic compounds. A compound can be determined to be therapeutic by screening, e.g., using the screening methods of the present invention. A “known therapeutic compound” refers to a therapeutic compound that has been shown (e.g., through animal trials or prior experience with administration to humans) to be effective in such treatment or prevention.
A compound is said to be “in a form suitable for administration” when the compound may be administered to an animal by any desired route (e.g., oral, intravenous, subcutaneous, intramuscular, etc.) and the compound or its active metabolites appear in the desired cells, tissue or organ of the animal in an active form.
As used herein, the term “therapeutic amount” refers to that amount of a compound required to neutralize undesirable pathologic effects in a subject.
As used herein, the term “C460W mutation” refers to the replacement of cysteine residue with a tryptophan residue at position 460 of the amino acid sequence of the rat olfactory cyclic nucleotide-gated ion channel. Similar mutations are contemplated to be useful in the context of the present invention, including but not limited to C460F and C460Y substitutions. In fact, any mutation(s) which also serves to decrease the NO sensitivity of the channel and increase the cAMP sensitivity of the channel find use with the methods and compositions of the present invention. Decreased NO sensitivity of a mutant channel can be determined by examining channel function in the presence and absence of a NO donor such as S-nitrosocysteine using published methods (Broillet, J. Biol. Chem., 275:15135-15141 [2000]), and increased cAMP sensitivity of a mutant channel can be determined by generating dose response curves for cAMP using the methods disclosed herein and in Rich et al. (J. Gen. Physiol., 118:63-77 [2001]).
As used herein, the term “E583M mutation” refers to the replacement of the glutamic acid residue with a methionine residue at position 583 of the amino acid sequence of the rat olfactory cyclic nucleotide-gated ion channel. Similar mutations are contemplated to be useful in the context of the present invention, including but not limited to E583V, E583L, and E583 I substitutions. In fact, any mutation(s), which also serves to increase cAMP sensitivity and/or decrease cGMP sensitivity finds use with the methods and compositions of the present invention. Increased cAMP sensitivity and/or decreased cGMIP sensitivity of a mutant channel can be determined by generating dose response curves for cAMP and cGMP, respectively using the methods disclosed herein and in Rich et al. (J. Gen. Physiol., 118:63-77 [2001]).
As used herein, the term “Δ61-90 mutation” refers to the deletion of residues 61-90 of the amino acid sequence of the rat olfactory cyclic nucleotide-gated ion channel. Other deletion(s), insertion(s) or substitution(s) which decrease the sensitivity of the channel to Ca2+-CaM regulation find use with the methods and compositions of the present invention. Decreased Ca2+-CaM sensitivity of a mutant channel can be determined by examining binding of radiolabeled CaM to mutant channels and measuring electric currents through the mutant channel in the presence of Ca2+-CaM using published methods (Liu et al., Science, 266:1348-1354 [1994]).
Importantly, channels with mutations in regions outside of those disclosed herein, are also contemplated to find use with the tools and techniques of the present invention
The term “stimulus” as used herein refers to any substance or agent which excites or produces a temporary increase of vital action in either in a whole cell or in any of its parts. Preferred “stimuli” are those which directly or indirectly alter cAMP levels in a cell or in a compartment of a cell.
The term “adenylate cyclase activator” as used herein, refers to compounds such as forskolin which are capable of activating the adenylate cyclase system and the biosynthesis of cAMP. “Forskolin” is derived from the plant coleus forskohlii.
The term “prostaglandin” as used herein, refers to any of any of a group of components derived from unsaturated 20 carbon fatty acids, primarily arachidonic acid, via the cyclooxygenase pathway that are extremely potent mediators of a diverse group of physiologic processes. The abbreviation for prostaglandin is PG, specific compounds are designated by adding one of the letters A through I to indicate the type of substituents found on the hydrocarbon skeleton and a subscript (1, 2 or 3) to indicate the number of double bonds in the hydrocarbon skeleton for example, PGE2. The predominant naturally occurring prostaglandins all have two double bonds and are synthesised from arachidonic acid (5, 8, 11, 14 eicosatetraenoic acid). The 1 series and 3 series are produced by the same pathway with fatty acids having one fewer double bond (8, 11, 14 eicosatrienoic acid) or one more double bond (5, 8, 11, 14, 17 eicosapentaenoic acid) than arachidonic acid. All of the prostaglandins act by binding to specific cell surface receptors causing an increase in the level of the intracellular second messenger cAMP (and in some cases cyclic GMP also).
As used herein, the terms “G-protein” and “GTP protein” refer to any one of a group of intracellular membrane associated proteins with a high affinity for guanine nucleotides, and which serve as second messengers or transducers of the receptor-initiated response to intracellular elements such as enzymes to initiate an effect. They are also mediators of activated cell-surface receptors and their enzymes, or of ion channels. G-proteins are responsible for activating a chain of events that alter the concentration of intracellular signaling molecules such as cAMP and calcium. Thus, the term “G-protein activator” refers to compounds such as cholera toxin, which are capable of activating G-proteins.
The term “phosphodiesterase” as used herein refers to an enzyme that cleaves phosphodiesters to give a phosphomonoester and a free hydroxyl group. In preferred embodiments, the term is used to refer to enzymes such as cAMP phosphodiesterase, that convert cyclic nucleotides to the monoester forms. Thus, the term “phosphodiesterase inhibitor” refers to compounds such as rolipram, which inhibit or antagonize the biosynthesis or actions of a phosphodiesterase.
As used herein, the term “dose response curve” refers to a graph depicting the relationship between the dose of a drug or other chemical (e.g., cAMP) and the degree of response it produces (e.g., electric current).
The term “electric current” as used herein refers to the rate of charge flow past a given point in an electric circuit, measured in amperes (coulombs/second).
As used herein, the term “fluorescent Ca2+ indicator” refers to compounds used to probe Ca2+ concentration via their fluorescent spectral changes upon Ca2+ binding. Fluorescent calcium indicators finding use with the methods and compositions of the present invention include but are not limited to Fura-2, Indo-1, Fluo-3 and Rhod-2.
The term “local intracellular cAMP concentration” refers to the cAMP concentration detected at a restricted or limited part of the cell (e.g., membrane localized). In contrast, the term “total intracellular cAMP concentration” refers to the cAMP concentration detected throughout the cell (e.g., entire accumulated amount divided by the accessible volume of the cell).
Experimental
The following examples are provided in order to demonstrate and further illustrate certain preferred embodiments and aspects of the present invention and are not to be construed as limiting the scope thereof.
In the experimental disclosure which follows, the following abbreviations apply: C (degrees Centigrade); rpm (revolutions per minute); BSA (bovine serum albumin); CFA (complete Freund's adjuvant); IFA (incomplete Freund's adjuvant); IgG (immunoglobulin G); IM (intramuscular); IP (intraperitoneal); IV (intravenous or intravascular); SC (subcutaneous); H2O (water); HCl (hydrochloric acid); aa (amino acid); bp (base pair); kb (kilobase pair); kD (kilodaltons); gm (grams); μg (micrograms); mg (milligrams); ng (nanograms); μl (microliters); ml (milliliters); mm (millimeters); nm (nanometers); μm (micrometer); M (molar); mM (millimolar); μM (micromolar); U (units); V (volts); MW (molecular weight); sec (seconds); min(s) (minute/minutes); hr(s) (hour/hours); w/v (weight to volume); v/v (volume to volume); MgCl2 (magnesium chloride); NaCl (sodium chloride); NO (nitric oxide); OD280 (optical density at 280 nm); OD600 (optical density at 600 nm); AC (adenylyl cyclase); AKAP (A-kinase anchoring protein); HEK (human embryonic kidney); NPE-cAMP (1-(2-nitrophenyl)ethyl-cAMP); oCNG channel (olfactory cyclic nucleotide-gated channel); PDE (phosphodiesterase); CaM (calmodulin); IBMX (3-isobutyl-1-methylxanthine); pCPT-cGMP (8-p-chlorophenylthio-cGMP; RO-20-1724 (4-(3-butoxy-4-methoxybenzyl)-2-imidazolidinone); VIP (vasoactive intestinal peptide); WT (wild-type); Tris (tris(hydroxymethyl) aminomethane); SDS (sodium dodecyl sulfate); PAGE (polyacrylamide gel electrophoresis); SDS PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis); PBS (phosphate buffered saline [150 mM NaCl, 10 mM sodium phosphate buffer, pH 7.2]); PEG (polyethylene glycol); PMSF (phenylmethylsulfonyl fluoride); PCR (polymerase chain reaction); RT-PCR (reverse transcription PCR); Molecular Probes (Molecular Probes, Eugene, Oreg.); Calbiochem (Calbiochem-Novabiochem Corp., San Diego, Calif.); Amersham (Amersham Pharmacia Biotech, Piscataway, N.J.); ICN (ICN Pharmaceuticals, Inc., Costa Mesa, Calif.); ATCC (American Type Culture Collection, Rockville, Md.); BioRad (BioRad, Richmond, Calif.); Clontech (CLONTECH Laboratories, Palo Alto, Calif.); Life Technologies, GIBCO BRL, and Gibco BRL (Life Technologies, Inc., Gaithersburg, Md.); Invitrogen (Invitrogen Corp., San Diego, Calif.); New England Biolabs (New England Biolabs, Inc., Beverly, Mass.); Novagen (Novagen, Inc., Madison, Wis.); Sigma (Sigma Aldrich, St. Louis, Mo.); Stratagene (Stratagene Cloning Systems, La Jolla, Calif.); Gemini (Gemini Bioproducts, Calabassas, Calif.); and Hi-Tech (Hi-Tech, Salisbury, United Kingdom). Mathworks (Mathworks, Natick, Mass.); Axon Instruments (Axon Instruments, Foster City, Calif.); Perkin-Elmer (Norwalk, Conn.); and Warner Instruments (Warner Instruments (Warner Instruments, Hamden, Conn.).
Data in the accompanying Figures are representative of at least four experiments. Unless otherwise indicated, all experiments were performed at room temperature (19-22° C.). Forskolin and PDE inhibitors were from Calbiochem. Fura-2/AM and pluronic F-127 were from Molecular Probes. [2-3H]Adenine, [3H]cAMP, and [α-32P]ATP were from Amersham. All other chemicals were from Sigma.
Human embryonic kidney (HEK-293) cells were maintained in culture and infected with adenovirus as known in the art (See, Rich et al. [2001], supra). As described in greater detail in Example 2, an adenovirus encoding the α subunit of the rat olfactory CNG channel (CNG2, CNC 3) with mutations C460W and E583M was constructed using the Quik Change Site-Directed Mutagenesis Kit (Stratagene) and a modification of the AdEasy system (He et al., Proc. Natl. Acad. Sci. USA 95:2509-2514 [1998]; and Orlicky and Schaack, J. Lipid Res., 42:460-466 [2001]). All cells used in cAMP assays were treated with this adenovirus.
Briefly, HEK-293 were maintained in MEM (Life Technologies Inc.) supplemented with 26.2 mM NaHCO3, 10% (v/v) fetal bovine serum (Gemini), penicillin (50 μg/mL), and streptomycin (501 g/mL), pH 7.0, at 37° C. in a humidified atmosphere of 95% air and 5% CO2. Cells were plated at ˜60% confluence in 100 mm culture dishes twenty-four hours prior to infection with the CNG-channel-encoding adenovirus constructs (multiplicity of infection=10 plaque forming units per cell). Two hours post-infection, hydroxyurea was added to the cell media at 2 mM final concentration to partially inhibit viral replication. Twenty-four hours post-infection cells were detached with phosphate-buffered saline containing 0.03% EDTA, resuspended in serum-containing medium, and assayed within 12 hours.
GH4C1 rat pituitary cells (ATCC) were maintained in 13 mL Ham's F-10 medium (Life Technologies) supplemented with 14.3 mM NaHCO3, 15% donor horse serum (Gemini), and 2.5% fetal bovine serum, pH 6.8, in 75 cm2 flasks at 37° C. in a humidified atmosphere of 95% air and 5% CO2. Cells were split weekly (1:4) and washed with fresh medium twice weekly. Cells were plated at ˜60% confluence in 100 mm culture dishes twenty-four hours prior to infection with the CNG-channel-encoding adenovirus constructs (multiplicity of infection=50 plaque forming units per cell). Forty-eight hours post-infection cells were detached, resuspended in serum-containing medium, and assayed within 12 hours.
In this Example, the methods used to construct CNG-channel encoding adenoviruses are described. As indicated above in Example 1, point mutations were introduced into the α subunit of the WT rat olfactory CNG channel (CNG2, CNC 3) using the QuikChange Site-Directed Mutagenesis Kit (Stratagene). Overlap extension PCR was used to delete the Ca2+-CaM binding site (amino acids 61-90). A replication-defective adenovirus, in which the channel coding sequence containing the E583M mutation replaced the E1 region, was constructed as known in the art and described previously (Fagan et al., J. Biol. Chem., 274:12445-12453 [2000]). Briefly, the cDNA encoding the mutant channel was ligated into the plasmid pACCMV (See, Gomez-Foix et al., J. Biol. Chem., 267:25129-25134 [1992]) under the control of the cytomegalovirus major immediate early (CMV) promoter between the BamHI and SalI sites. The plasmid was then digested with SalI and ligated with a BstBI adaptor. The resultant plasmid was then digested with BstBI and XmnI and ligated with BstBI-digested Ad5dl327Bst-gal-terminal-protein complex, that had been isolated by banding purified Ad5dl327Bst-gal (Schaack et al., J. Virol., 69:3920-3923 [1995]) virions in 2.8 M CsCl, 4 M guanidine-HCl. The ligation products were used to transfect HEK-293 cells using Ca3(PO4)2 precipitation (Jordan et al., Nucl. Acids Res., 24:596-601 [1996]).
The transfected cells were incubated for seven days. The cells were frozen and thawed to release virus, and dilutions used to infect HEK-293 plates for plaque purification. The infected HEK-293 plates were overlaid with medium in Noble agar, fed after 4 days, and stained with X-gal (5-bromo-4-chloro-3-indolyl-D-galactopyranoside) and neutral red. Clear plaques, which were derived from viral chromosomes lacking the LacZ gene of the parental virus, were amplified and analyzed by PCR and restriction digestion for the presence of the mutated CNG channel cDNA.
Adenovirus transducing vectors encoding C460W/E583M and Δ61-90/C460W/E583M channels were constructed using the AdEasy system (He et al., Proc. Natl. Acad. Sci. USA 95:2509-2514 [1998]). The cDNA encoding the mutant channel was ligated into pShuttle-CMV between the KpnI and XbaI sites. The resultant plasmid was then linearized by digestion with PmeI and used to transform E. coli strain BJ5183 that had been transformed with pAdEasy-1. A plasmid containing the adenovirus chromosome encoding the mutated CNG channel was digested with PacI to release the adenovirus chromosome, and this DNA was used to transfect HEK-293 cells. After incubation for seven days, the virus was released by freezing and thawing, and plaque purified. The purified virus was tested for the presence of the CNG channel cDNA by PCR. A virus containing the channel cDNA was grown in large scale in HEK-293 cells and purified by banding using CsCl step and isopycnic gradients.
To assess the cyclic nucleotide sensitivity of different CNG channel constructs, excised, inside-out patch recordings were made at room temperature (20-21° C.) using an Axopatch-200A patch clamp amplifier (Axon Instruments Inc.). Pipettes were pulled from borosilicate glass and heat polished. Pipettes were lowered onto the cells and gigaohm seals were formed. Patches were excised by shearing cells from the pipette with a jet of liquid. Ionic currents were elicited by 250 ms pulses to membrane potentials of +50 and −50 mV from a holding potential of 0 mV. Current records were sampled at five times the filter setting and stored on an IBM compatible computer. Records were corrected for errors due to series resistance (pipette resistance was 4.1 ±0.1 MΩ). Both the pipette and bath solutions contained (mM): 130 NaCl, 2 HEPES, 0.02 EDTA, and 1 EGTA, pH 7.6. Cyclic nucleotide-induced currents were obtained from the difference between currents in the presence and absence of cyclic nucleotides. Dose response curves for cAMP and cGMP were obtained at +50 and −50 mV in the same patch. The effects of the modifications were assessed using the Hill equation, I/Imax=[cNMP]N/([cNMP]N+K1/2N), where I/Imax is the fraction of maximal current, cNMP represents cyclic nucleotide, K1/2 is the concentration that gives half-maximal current, and N is the Hill coefficient, an index of cooperativity. K1/2 and N for cAMP were 36±5 μM, 10±2 μM, 1.0±0.3 μM, and 11±μM for WT, E583M, C460/E583M, and Δ61-90/C460W/E583M channels, respectively (See, Rich et al., J. Gen. Physiol. [2001], supra).
Increases in local cAMP concentration activate CNG channels and trigger Ca2+ entry. Ca2+ influx was monitored in cell populations using the fluorescent indicator fura-2. Cells were loaded with 4 μM fura-2/AM (the membrane permeant form) and 0.02% pluronic F-127 for 30-40 min, in a buffer containing Ham's F-10 medium supplemented with 1 mg/mL BSA and 20 mM HEPES, pH 7.4. In some experiments, HEK-293 cells were loaded with a higher concentration of fura-2/AM (16 μM) for comparison.
Cells were washed twice, resuspended in the buffer described above (3-4×106 cells/3 mL buffer solution), resuspended in a solution containing (mM): 145 NaCl, 11 D-Glucose, 10 HEPES, 4 KCl, 1 CaCl2, 1 MgCl2, and 1 mg/mL BSA, pH 7.4 (3-4×106 cells/3 mL buffer solution), and assayed using an LS-50B spectrofluorimeter (Perkin Elmer). Additions were made by pipetting stock solutions into a stirred cuvette. The solutions were then assayed using an LS-50B spectrofluorimeter (Perkin Elmer). Additions were made by pipetting stock solutions into a stirred cuvette (mixing time ˜5 s).
Fluorescence was measured at an excitation wavelength of 380 nm and an emission wavelength of 510 nm. Under these conditions Ca2+ influx was found to cause a decrease in fluorescence (ΔF), which was expressed relative to the pre-stimulus fluorescence (F0) to correct for variations in dye concentration, and to allow for comparison of results on different batches of cells. ΔF/F0 was plotted with inverted polarity, so that increases in Ca2+ influx were represented as positive deflections. Linear fits to the steady-state Ca2+ influx rates were used to quantify the results (Rich et al. [2001], supra). The MATLAB software package (MathWorks) was used for curve fitting and simulations.
Data were sampled at 0.5 Hz and filtered at 0.1 Hz (See
The measurement of absolute Ca2+ influx rates using fluorescence requires that fura-2 overwhelm endogenous Ca2+ buffers, and that the relation between fluorescence changes and Ca2+ entry be known (Schneggenburger et al., [1993]; Frings et al., [1995]). However, in these experiments, the concern was the relative Ca2+ influx rates, which report changes in cAMP levels. This requires only that fura-2 detect a fixed proportion of the entering Ca2+ in a given experiment. For this to be true, the concentration of unbound fura-2 should not change appreciably upon Ca2+ binding, and cellular Ca2+ buffers that are not overwhelmed by fura-2 should also not be significantly depleted by Ca2+ binding. Under these conditions, the equilibrium concentration of Ca2+-bound fura-2 (CaF), monitored by fluorescence at 380 nm, is given by:
where F represents fura-2, KF the dissociation constant for Ca2+ binding to fura-2, B the endogenous buffer, KB the dissociation constant for Ca2+ binding to the endogenous buffer, and CaT the total Ca2+ concentration that freely exchanges between fura-2 and the endogenous buffer. The equilibrium assumption is reasonable given the time scale of the experiments presented here (tens to hundreds of seconds) and the time scale of binding and unbinding of Ca2+ from fura-2 and endogenous buffers (milliseconds). Eq. 6 indicates that with [F] and [B] unchanged by the binding of Ca2+, [CaF] will be a constant fraction of [CaT], and therefore directly proportional to the amount of entering Ca2+. In experiments in which quantitative information was extracted (e.g., the in vivo estimate of KI, for PDE inhibitors), low levels of Ca2+ influx were purposely utilized.
Results from a typical experiment are shown in
There are several lines of evidence that free fura-2 levels did not change significantly at these low influx rates, and that depletion of endogenous buffers did not distort the measurements. First, after a brief delay (during which cAMP rose to steady level), the Ca2+ influx rate was constant for a substantial period (linear fit in
Neher and Augustine (Nether and Augustine [1992]) have shown in adrenal chromaffin cells that 98-99% of entering Ca2+ binds to endogenous buffers that are present at high concentration (375-750 μM) but have a low affinity for Ca2+ (KB˜5-10 μM). Thus, the ratio [B]/KB in Equation 6 was estimated to be 75. KF for fura-2 was estimated to be 150 nM. Thus, the [F]/KF values under the two loading conditions were expected to be 233 and 933. At the higher loading condition, fura-2 may be overwhelming the low-affinity cellular buffers. However, as pointed out above, this is not necessary as long as the buffers were not being depleted. It is very likely that low affinity buffers are present at a high enough concentration that they were not depleted by the low amounts of entering Ca2+. Two of the lines of evidence cited above indicate that buffers (including any high-affinity buffers that may be present) did not affect the proportion of Ca2+ detected by fura-2: the linearity of the influx traces (
There are over 30 known forms of PDE that have been grouped into 11 families. Of these families, PDE types V, VI, and IX are cGMP-specific. Recently, an isoform of PDE type IV, PDE4A, purified from U937 monocytic cells was shown to be inhibited by rolipram with an IC50 of 3 nM (MacKenzie and Houslay Biochem. J., 347:571-578 [2000]). It should be noted that it is contemplated that these efficacies may be modulated in vivo. For example, it has been shown that forms of PDE type IVA are approximately 10-fold more sensitive to rolipram when they bind to the SRC family tyrosyl kinase LYN (McPhee et al., [1999]). Data in Table 3 were obtained using heterologously expressed mouse (types VII, VIII, and X) and human (types I, II, IV, V, VII, IX, and XI) PDEs, as well as endogenous PDEs from dog kidney (type IV), rat brain (type IV), bovine heart (types III and IV), human heart (type II), bovine aorta (types I, III, and V), rabbit aorta (types I, IV, and V), and bovine photoreceptor (type VI). Data in Table 3 were compiled from: (1) Ahluawalia and Rhoads Biochem. Pharmacol., 31:665-669 (1982); (2) Ahn et al., Biochem. Pharmacol., 38:3331-3339 (1989); (3) Beavo, Adv. Second Messenger Phosphoprot. Res., 22:1-38 (1988); (4) Bolger et al., Mol. Cell. Biol., 13:6558-6571 (1993); (5) Bolger et al. Biochem. J., 328:539-548 (1997); (6) Coste and Grondin, Biochem. Pharmacol., 50:1577-1585 (1995); (7) Epstein et al., Arch. Biochem. Biophys., 218:119-133 (1982); (8) Fawcett et al., Proc. Natl. Acad. Sci. USA 97:3702-3707 (2000); (9) Fisher et al., J. Biol. Chem., 273:15559-15564 (1998); (10) Gardner et al., Biochem. Biophys. Res. Commun., 272:186-192 (2000); (11) Harrison et al., Meth. Enzymol., 15:685-702 (1988); (12) Hetman et al., Proc. Natl. Acad. Sci. USA 97:472-476 (2000); (13) Holck et al., J. Cardiovasc. Pharm., 6:520-530 (1984); (14) Lorenz and Wells, Mol. Pharmacol., 23:424-430 (1983); (15) Loughney et al., J. Biol. Chem., 271:796-806 (1996); (16) Loughney et al., Gene 216:139-147 (1998); (17) Nemoz et al., Biochem. Pharmacol., 15:2997-3000 (1985); (18) Podzuweit et al., Cell Signal 7:733-738 (1995); (19) Rosman et al., Gene 191:89-95 (1997); (20) Soderling et al., Proc. Natl. Acad. Sci. USA 95:8991-8996 (1998a); (21) Soderling et al., J. Biol. Chem., 273:15553-15558 (1998b); (22) Soderling et al., Proc. Natl. Acad. Sci., 96:7071-7076 (1999); and (23) Whalin et al., Mol. Pharmacol., 39:711-717 (1991). References cited for various inhibitors include: IBMX3,6,8,10-12,15,16,20-22, 8-M-IBBX2,4,14,15, EHNA12,16,18-22, Trequinsin23, Quazinone13, Etazolate1, RO-20-17247,9,12,20,21,23, Rolipram5,8,9,15-17,20-23, and Zaprinast6,8,9,15,16,20-22. Blank spaces in Table 3 indicate that these data are not available.
The PDE type(s) that regulates cAMP levels near CNG channels in HEK-293 were identified cells using the Ca2+ influx assay in cell populations as described in EXAMPLE 3. The C460W/E583M channels were used to monitor forskolin-stimulated cAMP accumulation in the presence and absence of a series of PDE inhibitors. The reported IC50 Values of inhibitors used in this experiment for each PDE type are given in Table 3. The PDE inhibitor concentrations used were typically at least 5-fold higher than the most potent IC50. Either vehicle or PDE inhibitors were added at 0 s and 1 μM forskolin was added at 180 s (See,
To further establish that PDE type IV is responsible for the observed IBMX-sensitive PDE activity, the KI's (inhibition constants) of IBMX and two PDE type IV inhibitors, RO-20-1724 and rolipram were estimated in vivo. This required developing a quantitative framework to assess the relationship between cAMP synthesis, hydrolysis, and redistribution throughout the cell. Thus, the following formalism was adopted:
where C is the steady-state rate of cAMP synthesis by AC, Vmax is the maximal rate of cAMP hydrolysis, Km is the Michaelis constant for PDE, and kf is the rate constant of cAMP flux out of the microdomain. In order to estimate KI for the PDE inhibitors, two assumptions were made. First, it was assumed that at low levels of AC stimulation and PDE inhibition, the concentration of local cAMP is low and diffusion of cAMP out of the microdomain is negligible. Second, it was assumed that cAMP levels reach steady-state shortly after AC stimulation (i.e., equal rates of synthesis and hydrolysis). With these assumptions, Equation 4 can be simplified to:
Interestingly, this equation reveals that when the inhibitor concentration is equal to KI, the cAMP concentration is twice that in the absence of inhibitor. At low cAMP concentrations (<K1/2 for the channel), the cAMP concentration is proportional to the square root of the Ca2+ influx rate (the Hill coefficient for channel activation is approximately 2). Thus, KI, can be estimated using a linear fit to the square root of the slopes of the fluorescence traces as a function of inhibitor concentration (See,
The high cAMP affinity C460W/E583M channels were used to detect changes in cAMP concentration following pretreatment with PDE inhibitors (added at 0 s) and modest forskolin stimulation (0.5 μM added at 180 s). IBMX, RO-20-1724, and rolipram were completely equilibrated across the plasma membrane of HEK-293 cells in <180 s (data not shown). The assumption that cAMP levels reach steady-state is supported by the long-lasting linear rise in Ca2+ concentration following forskolin stimulation (a steady Ca2+ influx rate reflects a constant cAMP level; See,
In this Example, experiments conducted to detect cAMP levels in cells are described. Changes in cyclic AMP concentrations in response to PGE1 stimulation were monitored in HEK-293 cells by measuring Ca2+ influx through C460W/E583M CNG channels (See, Rich et al. [2001], supra). HEK-293 cells express a variety of extracellular receptors including prostanoid receptors (Thomas and Hoffman, Mol. Pharmacol., 49:907-914 [1996]), and the cAMP-specific PDE type IV (Rich et al. [2001], supra; and Hoffmann et al., EMBO J., 18:893-903 [1999]). Based on PCR analysis, these cells also appear to express AC types II, II, VI, and VII, (Hellevuo et al., Biochem. Biophys. Res. Commun., 192:311-318 [1993]). C460W/E583M channels were expressed heterologously using an adenovirus construct (See, Rich et al. [2001], supra). The Ca2+ permeability of the channel was used to detect changes in local cAMP concentration; the fluorescent indicator fura-2 was used to monitor Ca2+ entry. With this approach, incremental changes in cAMP concentration are readily detected as changes in relative Ca2+ influx rates through C460W/E583M channels (See, Rich et al., [2001], supra).
The basis for the transient response was investigated further, as shown in
In the absence of PGE1, PDE inhibitors caused no measurable Ca2+ influx. These experiments suggest that AC activity did not decrease appreciably, but instead that PDE activity was up-regulated. To test whether PGE1 did indeed increase the total PDE activity, 100 nM PGE1 were added at various times after the initial application of 100 nM PGE1 (a subsaturating concentration). There was a negligible response to the subsequent addition of PGE1, even after 50 min (
In this Example, experiments conducted to examine the response of excitable GH4C1 pituitary cells to stimulation by either forskolin or the hormone vasoactive intestinal peptide (VIP), in the presence of absence of PDE inhibitors are described. In the experiments described, 1 μM nimodipine was added at time zero to block Ca2+ influx though voltage-gated Ca2+ channels (triggered by membrane depolarization due to Ca2+ and Na+ influx through CNG channels). This concentration of nimodipine was sufficient to block Ca2+ influx through voltage-gated Ca2+ channels activated by membrane depolarization in 24 mM external KCl, and did not alter forskolin or pCPT-cGMP induced Ca2+ influx through CNG channels expressed in HEK-293 cells (data not shown).
Next, forskolin-induced responses and the effects of IBMX in cells expressing the high cAMP affinity construct, C460W/E583M were examined. In these experiments either vehicle or 100 μM IBMX was added at 60 s (See,
Single-cell cAMP measurements were made using either the perforated patch or whole cell patch clamp technique (Rich et al. [2000], supra). In the perforated patch configuration, the pore forming antibiotic nystatin was added to the pipette solution to gain electrical access to the cell's interior while retaining divalent cations and larger molecules like cAMP in the cell. Recordings were made using an Axopatch-200A patch clamp amplifier (Axon Instruments). Pipette resistance was limited to 5 ΩM and averaged 3.4±0.5 ΩM (measurements provided herein are expressed as mean±standard deviation). In the perforated patch configuration, a steady access resistance was obtained 5-15 minutes following seal formation. Capacitive transients were elicited by applying −30 mV steps from the holding potential of −20 mV for calculation of access resistance (100±40 MΩ). These quantities were monitored throughout the experiments to ensure stable electrical access was maintained. Current records were typically sampled at five times the filter setting. Records were digitally filtered at 12 Hz, resampled at 60 Hz, and corrected for errors due to series resistance. The control bath solution contained (mM): 140 NaCl, 4 KCl, 11 glucose, 10 HEPES, and either 0.1 or 10 MgCl2, pH 7.4. Solutions were applied using the SF-77B fast-step solution switcher (Warner Instruments). The mechanical switch time was 1 ms. The time to exchange the extracellular solution was measured by applying a 140 mM KCl solution to a depolarized cell (+50 mV), and monitoring changes in current through endogenous voltage-gated K+ channels; for each experiment, it was less than 60 ms. The pipette solution in perforated patch experiments contained 70 mM KCl, 70 mM potassium gluconate, 4 mM NaCl, 0.5 mM MgCl2, 10 mM HEPES, pH 7.4, and 50-200 μg/ml nystatin. In most of these experiments, the pipette solution also contained 1 mM cAMP; at the end of the experiment the maximal cAMP-induced current could be measured by rupturing the cell membrane at the tip of the pipette with suction, allowing saturating cAMP to diffuse to the channels (See, Rich et al. [2000], supra). In whole cell experiments, 5 mM K2ATP and 0.1 mM Na2GTP were added to the pipette solution, and nystatin was not included.
The cyclic nucleotide sensitivity of the C460W/E583M CNG channel was assessed in excised, inside-out patches as known in the art (See, Rich et al. [2001], supra). K1/2 and N were 1.1±0.3 μM and 2.1±0.4 at +50 mV; and 1.0±0.3 μM and 2.0 ±0.3 at −50 mV (n=11). With these values, the cAMP concentration could be calculated from currents measured in perforated patch experiments (Rich et al. Proc. Natl. Acad. Sci. [2001], supra). For example, if I/Imax was found to be 0.6, the estimated cAMP concentration would be 1.2 μM. It should be noted that the low concentration of channels expressed in these cells (˜1 nM) did not significantly buffer the measured cAMP signals.
The response to PGE1 in single cells was examined by directly measuring ionic currents through C460W/E583M channels with the perforated patch clamp technique. This approach has higher temporal resolution and dynamic range than the Ca2+ influx assay, and the response can be calibrated, allowing for accurate measurement of cAMP concentration. These experiments were done in nominally Ca2+-free solutions, which increased currents through the channels (Ca2+ is a permeant blocker), and removed the possibility that Ca2+ entry inhibited the channels (See, Finn et al., Ann. Rev. Physiol., 58:395-426 [1996]). The responses of two different cells to rapid application of 1 μM PGE1 are shown in
All publications and patents mentioned in the above Specification are herein incorporated by reference. Various modifications and variations of the described method and system of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention which are obvious to those skilled in molecular biology, genetics, and/or related fields are intended to be within the scope of the present invention.
This application is a divisional of U.S. application Ser. No. 10/295,573, filed on Nov. 15, 2002 now U.S. Pat. No. 7,166,463, which claims benefit of provisional U.S. application Ser. No. 60/332,494, filed on Nov. 16, 2001.
This invention was made with government support under grants GM32438, NS28389, HL58344, DC00385, EY09275, from the National Institutes of Health. The United States Government has certain rights in the invention.
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| 2255548 | Jun 2000 | CA |
| WO 9858074 | Dec 1998 | WO |
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| Number | Date | Country | |
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| 20070099226 A1 | May 2007 | US |
| Number | Date | Country | |
|---|---|---|---|
| 60332494 | Nov 2001 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 10295573 | Nov 2002 | US |
| Child | 11601408 | US |
