Patent Application
20190094204
This document relates to materials and methods for a membrane transporter assay (e.g., an oscillating stimulus transporter assay (OSTA)). OSTA can be used to obtain temporal readouts of transporter activity. For example, OSTA can be used to screen for membrane transporter modulators (e.g., agonists or antagonists) having particular pathophysiological interest. This document also provides materials and methods for modulating membrane transporter function. For example, modulators of membrane transporters (e.g., drugs and other stimuli) can be used to treat membrane transporter associated diseases and/or disorders (e.g., cystic fibrosis, stroke, epilepsy, Alzheimer's disease, Huntington's disease, and amyotrophic lateral sclerosis).
Membrane transporter proteins enable the passage of nearly all biologically relevant molecules across the otherwise-impermeable lipid membranes of cells and organelles, and as such, are of great importance to life. Genes encoding membrane transporter proteins constitute >10% of the human genome, and mutations thereof are implicated in a wide range of human genetic diseases and cancers (Sahoo et al., 2014 Front. Physiol. 5:91). Despite this importance, methods for functional measurement have been limited. Transporter proteins are embedded in lipid membranes making purification and characterization of active transporters difficult or even impossible.
This document provides materials and methods for a membrane transporter assay (e.g., an oscillating stimulus transporter assay (OSTA)). OSTAs can be used to obtain readouts of transporter activity such as temporal readouts. In some cases, OSTAs can be used to screen for modulators of a membrane transporter. This document also provides materials and methods for modulating membrane transporter function. For example, modulators (e.g., agonists or antagonists) of membrane transporter can be used to treat membrane transporter associated diseases and/or disorders (e.g., cystic fibrosis, stroke, epilepsy, Alzheimer's disease, Huntington's disease, amyotrophic lateral sclerosis (ALS)).
As demonstrated herein, OSTA can be used to monitor (e.g., characterize and/or analyze) the function of membrane transporters embedded in cellular membranes of living cells. OSTA can be used to characterize conditions under which a membrane transporter (e.g., SLC26a3 and SLC26a2) transports most effectively. OSTA can be used to characterize membrane transporter modulator kinetics (e.g., inhibition kinetics) and analyze responsiveness of a membrane transporter to one or more membrane transporter modulators. Thus, OSTAs enable facile and rapid measurement of a wide variety of transporter properties.
In general, one aspect of this document features a method of characterizing membrane transporter function (e.g., transport rate, transporter activity, and transporter inhibition). The method includes, or consists essentially of, expressing a sensor in a cell comprising a membrane transporter, wherein the sensor capable of detecting a substrate transported by the membrane transporter; perfusing the cell with a solution including the substrate, wherein said solution changes or oscillates between a low substrate concentration and a high substrate concentration; and detecting the substrate, wherein changes in detection of the substrate can be used to characterize membrane transporter function. The membrane transporter can be an endogenous or exogenous membrane transporter. The membrane transporter can be any transporter for which a sensor exists to detect transporter activity either directly or indirectly, e.g., changes in substrate or co-substrate concentration, or changes in corresponding solution properties such as pH, which depend on transporter activity. Examples of transporters already successfully measured include, but are not limited to the Glut family, the Sglt family, the EAAT family, the SLC26 family, the NBC family, and sodium-alanine co-transporters. These transporter families may specifically include transporters such as glucose transporter 1 (GLUT1), GLUT2, SLC26a3, SLC26a2, Sglt2, EAAT2, or NBCe1-b.
In some aspects, the membrane transporter can be SLC26a3 and the membrane transporter substrate can include Cl— and HCO3-. In some aspects, the membrane transporter can be SLC26a2 and the membrane transporter substrate can include SO42- and OH—. In some aspects, the membrane transporter can be Sglt2 and the membrane transporter substrate can include Na+ and glucose. In some aspects, the membrane transporter can be EAAT2 and the membrane transporter substrate can include glutamate. In some aspects, the membrane transporter can be NBCe1-b and wherein the membrane transporter substrate can include Na+ and HCO3-. In some aspects, the membrane transporter can be Glut family transporters and the membrane transporter substrate can include glucose.
Expressing a sensor can include transfecting the cell with a nucleotide sequence encoding a peptide sensor. The sensor can be a fluorescent sensor. The fluorescent sensor can be a fluorescent Ca2+ sensor (e.g., Furas, OGB, Fluos, or Indos). The fluorescent sensor can be a fluorescent Mg2+ sensor (e.g., mag-Indo, mag-Fura, or mag-Fluo). The fluorescent sensor can be a fluorescent voltage sensor (e.g., di-ANNEPSs or PeTs). The fluorescent sensor can be a fluorescent H+ sensor (e.g., a SNARF dye, such as SNARF-5F-AM, or a BCECF dye. The fluorescent sensor can be a fluorescent glucose sensor (e.g.,“Sweetie” a fluorescent glucose biosensor). The fluorescent sensor can be a fluorescent glutamate sensor (e.g., the iGluSnFR protein). Other sensors may be for chloride, tryptophan, nicotine, alanine, glycine, proline, maltose, maltotriose, pH, nucleotides, cyclic nucleotides, dinucleotides, glutathione, or peptides. The cell can be any type of cell. The cell can be a mammalian cell. The cell can be an adherent cell. The cell can be a HEK-293 cell or an E. coli cell.
Perfusing the cell with a solution including the substrate can be a continuous perfusion. The oscillation period can be about 10-120 seconds. The solution can oscillate between about 0-5 M substrate concentration.
Detecting the substrate can include obtaining one or more images of the cell. A series of images of the cell can be obtained using a microscope. In some aspects, the sensor can be a fluorescent sensor and the microscope can be a fluorescence microscope. The series of images of the cell can be a time-lapse movie. The time-lapse movie can include frame rates of about 1-2 Hz.
In some embodiments, this document features a method of characterizing Cl—/HCO3- exchange. The method includes, or consists essentially of, expressing a fluorescent H+ sensor in a cell comprising a Cl—/HCO3- exchange transporter, wherein the fluorescent H+ sensor comprises a SNARF-5F-AM dye; perfusing the cell with a solution comprising Cl—, wherein said solution oscillates between about 8 mM Cl— and about 158 mM Cl—; and detecting the H+, wherein changes in the H+ can be used to characterize Cl—/HCO3- exchange across a cell membrane.
In some embodiments, this document features a method of characterizing SO42-/OH— exchange. The method includes, or consists essentially of, expressing a fluorescent H+ sensor in a cell comprising a SO42-/OH— exchange transporter, wherein the fluorescent H+ sensor comprises a SNARF-5F-AM dye; perfusing the cell with a solution comprising SO42-, wherein said solution oscillates between about 0 mM SO42- and about 100 mM SO42-; and detecting H+, wherein changes in the H+ can be used to characterize SO42-/OH— exchange.
In some embodiments, this document features a method of characterizing glucose transporter function. The method includes, or consists essentially of, expressing a fluorescent glucose sensor in a cell comprising a glucose transporter, wherein the fluorescent glucose sensor is a cytosolic ratiometric peptide; perfusing the cell with a solution comprising glucose, wherein said solution oscillates between about 0 mM glucose and about 2-100 mM glucose; and detecting the glucose, wherein changes in detection of the glucose can be used to characterize glucose transporter function. The glucose transporter can be a Na+-dependent glucose transporter function, and the perfusing step further can include perfusing the cell with a solution comprising Na+ and/or K+.
In some embodiments, this document features a method of characterizing glutamate transporter function. The method includes, or consists essentially of, expressing a fluorescent glutamate sensor in a cell comprising a glutamate transporter, wherein the fluorescent glutamate sensor comprises iGluSnFR; perfusing the cell with a solution comprising glutamate, wherein said solution oscillates between about 0 mM glutamate and about 10 mM glutamate; and detecting the glutamate, wherein changes in detection of the glutamate can be used to characterize glutamate transporter function.
In another aspect, this document features a method of identifying a substrate of a membrane transporter. The method includes, or consists essentially of, expressing a sensor in a cell comprising a membrane transporter, wherein the sensor can detect a candidate substrate; perfusing the cell with a solution comprising the candidate substrate, wherein said solution oscillates between a high candidate substrate concentration and a low candidate substrate concentration; and detecting transport of the candidate substrate, wherein transport of the candidate substrate identifies the candidate substrate as the substrate of the membrane transporter.
In another aspect, this document features a method of screening for modulators of membrane transporter function. The method includes, or consists essentially of, expressing a sensor in a cell comprising a membrane transporter, wherein the sensor can detect a substrate transported by the membrane transporter; perfusing the cell with a solution comprising the substrate, wherein said solution oscillates between a low substrate concentration and a high substrate concentration; providing the cell with a candidate modulator; and detecting transport of the substrate, wherein a change in transport of the substrate identifies the candidate modulator as a modulator of membrane transporter function.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Methods and materials are described herein for use in the present disclosure; other, suitable methods and materials known in the art can also be used. The materials, methods, and examples are illustrative only and not intended to be limiting. All publications, patent applications, patents, sequences, database entries, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control.
The details of one or more embodiments of the invention are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the invention will be apparent from the description and drawings, and from the claims.
DESCRIPTION OF THE DRAWINGS
This document provides materials and methods for a membrane transporter assay (e.g., an oscillating stimulus transporter assay (OSTA)). The membrane transporter assay described herein can be performed on membrane transporters in situ (e.g., embedded in the cellular membrane of living cells). A membrane transporter assay described herein can drive substrate transport and image the response of a sensor to measure transporter function. Further, not only are the methods provided herein are easier and faster both to implement and to run than current methods of studying membrane transporters, but functional imaging techniques provided herein achieve greater temporal resolution and statistical robustness. In some cases, a membrane transporter assay described herein can be used to monitor membrane transporter function (e.g., to monitor membrane transporter function as a function of time). For example, a membrane transporter assay described herein can be used to characterize and/or analyze membrane transporter function as a function of time. For example, OSTA can be used to obtain temporal readouts of transporter activity or to measure time-dependent responses to membrane transporter modulators (e.g., drugs and other stimuli). In some cases, OSTA can be used to screen for membrane transporter modulators (e.g., agonists or antagonists) having particular pathophysiological interest. This document also provides materials and methods for modulating membrane transporter function. For example, modulators of membrane transporter (e.g., drugs and other stimuli) can be used to treat membrane transporter associated diseases and/or disorders (e.g., cystic fibrosis, cerebral stroke, epilepsy, Alzheimer's disease, Huntington's disease, amyotrophic lateral sclerosis (ALS)).
A membrane transporter assay described herein can characterize and/or analyze membrane transporter function in any appropriate cell. A cell can have an endogenous membrane transporter or an exogenous membrane transporter. In cases where a cell has an exogenous membrane transporter, the membrane transporter can be a recombinant membrane transporter. A cell can be an adherent cell or a non-adherent cell. A cell can be from any appropriate source. For example, a cell that can be used in a membrane transporter assay described herein can from a mammal (e.g., human, mouse, hamster, dog, rabbit, primate, and cat), non-mammalian animal, a plant, a bacterium, a fungus (e.g., a yeast), and a protist (e.g., algae). A cell can be any appropriate cell type (e.g., a kidney cell, a neuron, a myocyte, an epithelial cell, a blood cell, and a liver cell). Examples of cells that can be used in a membrane transporter assay described herein include, without limitation, HEK-293, MDCKII, and Caco-2 cells. For example, a cell that can be used in a membrane transporter assay described herein to characterize and/or analyze membrane transporter function can be a HEK-293 cell.
A membrane transporter assay described herein can characterize and/or analyze any appropriate membrane transporter. A membrane transporter can be any membrane protein involved in the movement of a substrate across a lipid bilayer, a cell membrane and/or an organelle membrane. Transport across a cell membrane can be transport from the intracellular side of the cell membrane to the extracellular side of the cell membrane or from the extracellular side of the cell membrane to the intracellular side of the cell membrane. A membrane transporter can facilitate movement of a substrate across a cell membrane by passive transport (e.g., facilitated diffusion) or by active transport (e.g., transport of a substrate across a membrane against its concentration gradient). Active transport can be primary active transport (e.g., requiring chemical energy such as ATP) or secondary active transport (e.g., requiring an electrochemical gradient). A membrane transporter can be a carrier (e.g., allowing only a small amount of substrate to be transported) or a channel (e.g., allowing thousands to millions of substrate molecules, such as ions, to be transported). A membrane transporter can be a glutamate transporter (e.g., an excitatory amino acid transporter (EAAT) or a vesicular glutamate transporter (VGLUT)), an ion transporter (e.g., calcium (Ca2+) pumps, magnesium (Mg2+) transporters, sodium (Na+) transporters, and potassium (K+) transporters), a bicarbonate (HCO3-) transporter, or a sulfate (SO42-) transporter. In some cases, a membrane transporter can be an exchanger (e.g., an anion exchanger) that transports a first substrate (e.g., a first molecule or ion) and exchanges it for a second substrate (e.g., a second molecule or ion). A membrane transporter can be a recombinant membrane transporter.
A membrane transporter can be any of:
1: Channels/pores
2: Electrochemical potential-driven transporters
3: Primary active transporters
4: Group translocators
The group translocators provide a special mechanism for the phosphorylation of sugars as they are transported into bacteria (PEP group translocation)
5: Electron carriers
The transmembrane electron transfer carriers in the membrane include two-electron carriers, such as the disulfide bond oxidoreductases (DsbB and DsbD in E. coli) as well as one-electron carriers such as NADPH oxidase. Often these redox proteins are not considered transport proteins.
A membrane transporter can be a member of a solute carrier family (SLC). A membrane transporter can be a glucose transporter (GLUT). Examples of membrane transporters that can be characterized and/or analyzed with a membrane transporter assay described herein include, without limitation, EAAT1 (SLC1A3), EAAT2 (GLT-1 or SLC1A2), EAAT3 (SLC1A1), EAAT4 (SLC1A6), EAAT5 (SLC1A7), VGLUT1 (SLC17A7), VGLUT2 (SLC17A6), VGLUT3 (SLC17A8), GLUT1 (SLC2A1), GLUT2, SLC26a3, SLC26a2, Sglt2, NBCe1-b, plasma membrane Ca2+ ATPase (PMCA), Na+/Ca2+ exchanger (NCX), MRS2, SLC41 (MgtE), TRPM6/TRPM7, and band 3 anion exchange proteins. In some cases, a membrane transporter can be GLUT1, GLUT2, SLC26a3, SLC26a2, Sglt2, EAAT2, or NBCe1-b. Examples of substrates that can be transported by a membrane transporter include, without limitation, ions (e.g., Ca2+, Mg2+, Na+, H+, Zn2+, and K+), HCO3−, OH−, SO42−, amino acids (e.g., glutamate), small molecules (e.g., glucose), and macromolecules (e.g., proteins, DNA, and RNA). In some cases, a membrane transporter described herein can transport a specific substrate. For example, in cases where the membrane transporter is SLC26a3, the substrate can be Cl− and/or HCO3−. For example, in cases where the membrane transporter is SLC26a2, the substrate can be SO42− and/or OH−. For example, in cases where the membrane transporter is Sglt2, the substrate can include Na+ and/or glucose. For example, in cases where the membrane transporter is EAAT2, the substrate can include glutamate. For example, in cases where the membrane transporter is NBCe1-b, the substrate can include Na+ and/or HCO3−.
A membrane transporter assay described herein can use any appropriate sensor. A sensor described herein can detect the presence of substrate. A sensor can indicate the presence of a substrate and may also be referred to herein as an indicator. The sensor can be any sensor capable of detecting a substrate transported by the membrane transporter. In some cases, a sensor can be an intracellular sensor. In some cases, a sensor can detect a specific substrate. For example, a sensor can detect an ion (e.g., Ca2+, Mg2+, Na+, H−, or Zn2+), HCO3−, OH−, SO42−, an amino acid (e.g., glutamate), a small molecule (e.g., glucose), or a macromolecule (e.g., a protein). In some cases, a substrate can be labeled, and a sensor can detect the label (e.g., chitin binding protein (CBP), maltose binding protein (MBP), glutathione-S-transferase (GST), poly(His) tag, thioredoxin (TRX), poly(NANP), FLAG-tag, V5-tag, Myc-tag, HA-tag, NE-tag, and biotin). A sensor can be any appropriate type of molecule. In some cases, a sensor can be a protein sensor. A sensor described herein can provide an output, such as a visual signal (e.g., fluorescence, colorimetric dyes, absorbance changes, and turbidity) or an electrical signal (e.g., conductivity and resistance). In some cases, a sensor can be a fluorescent sensor. For example, a sensor that can be used to detect the presence of a substrate in a membrane transporter assay described herein can be a fluorescent protein sensor. Examples of sensors that can be used to detect a substrate as described herein include, without limitation, Furas, OGB, Fluos, Indos, mag-Indo, mag-Fura, mag-Fluo, di-ANNEPSs, PeTs, SNARF dyes (e.g., SNARF-5F-AM), BCECF dyes, cytosolic ratiometric peptides, intensity-based glutamate-sensing fluorescent reporters (iGluSnFRs; see, e.g., WO 2013/052946, and Marvin et al., 2013 Nat Methods. 10:162-70). In some cases, a sensor described herein can detect a specific substrate. For example, in cases where the substrate is H+/pH, the sensor can be a SNARF or a BCECF. For example, in cases where the substrate is Ca2+, the sensor can be a Fura, a OGB, a Fluos, or a Indos. For example, in cases where the substrate is Mg2+, the sensor can be a mag-Indo, a mag-Fura, or a mag-Fluo. For example, in cases where the substrate is K+ (e.g., to detect transmembrane potential), the sensor can be a di-ANNEPSs or a PeTs. For example, in cases where the substrate is H+ (e.g., to detect pH changes associated with changes in, for example, OH− or HCO3−), the sensor can be a SNARF dye (e.g., SNARF-5F-AM) or a BCECF dye. For example, in cases where the substrate is glucose, the sensor can be a cytosolic ratiometric peptide. For example, in cases where the substrate is glutamate, the sensor can be an iGluSnFR. The sensor described herein can be provided to a cell by any appropriate means. For example, a sensor can be provided to a cell by transfection, transduction, or electroporation, into a cell. In some cases, a membrane transporter assay described herein can include expressing a nucleic acid sequence encoding a protein sensor in a cell having a membrane transporter described herein. A nucleic acid sequence encoding a protein sensor can be a recombinant nucleic acid sequence. A nucleic acid sequence encoding a protein sensor can be naked nucleic acid or can be in a vector (e.g., a plasmid vector, an expression vector, or a viral vector). For example, a sensor can be provided to a cell described herein by transfecting the cell with an expression vector including a nucleic acid sequence encoding a protein sensor. In some cases, a membrane transporter assay described herein can include loading a cell having a membrane transporter described herein with a small molecule sensor. For example, a sensor can be provided to a cell described herein by electroporating the cell with a small molecule sensor.
A membrane transporter assay described herein can include providing a cell described herein (e.g., having a membrane transporter and a sensor) with substrate. In some cases, a membrane transporter can be an exchanger (e.g., an anion exchanger), and a membrane transporter assay described herein can include providing a cell having a membrane transporter and a sensor with a first substrate and a second substrate. A substrate can be provided in a solution. In some cases, providing a cell described herein with substrate can include perfusion the cell with a substrate solution. The perfusion can be continuous or discontinuous. In some cases, a cell having a membrane transporter and a sensor with substrate can include continuously perfusion the cell with a substrate solution. A substrate solution can oscillate between having a high substrate concentration and having a low substrate concentration. For example, a solution having a high substrate concentration can have at least about 1 mM substrate (e.g., at least about 2, at least about 10, at least about 25, at least about 50, at least about 75, at least about 100, at least about 120, at least about 130, at least about 140, at least about 150, at least about 160, at least about 170, at least about 180, at least about 190, or at least about 200 mM). For example, a solution having a low substrate concentration can have no greater than about 0 mM substrate (e.g., greater than about 1, greater than about 2, greater than about 5, greater than about 8, greater than about 10, greater than about 15, or greater than about 25 mM). In some cases, a substrate solution can oscillate between about 0 and about 5 M substrate (e.g., can oscillate between about 0 mM and about 150 mM, can oscillate between about 0 mM and about 100 mM, can oscillate between about 0 mM and about 50 mM, can oscillate between about 0 mM and about 10 mM, can oscillate between about 0 mM and about 2 mM, can oscillate between about 5 mM and about 160 mM, can oscillate between about 8 mM and about 158 mM, can oscillate between about 10 mM and about 150 mM, or can oscillate between about 25 mM and about 150 mM substrate). For example, a substrate solution can oscillate between having about 8 mM and about 158 mM Cl−. For example, a substrate solution can oscillate between having about 0 mM and about 100 mM SO42−. For example, a substrate solution can oscillate between having about 0 mM and about 150 mM K+. For example, a substrate solution can oscillate between having about 25 mM and about 150 mM K+. For example, a substrate solution can oscillate between having about 0 mM and about 2 mM glucose. For example, a substrate solution can oscillate between having about 0 mM and about 10 mM glutamate (e.g., monosodium glutamate). In cases where a first substrate and a second substrate are provided, the first substrate and the second substrate can be oscillated in conjunction or in opposition. An oscillation period is the amount of time it takes to complete a full cycle of perfusion subjecting a cell to both a high substrate concentration and a low substrate concentration. For example, an oscillation period of 10 seconds would include perfusion a cell with a high substrate concentration of 5 seconds and perfusion a cell with a low substrate concentration for 5 seconds. In some cases, an oscillation period can be about 0.1 to about 600 seconds/period (e.g., about 10 to about 120, about 15 to about 100, about 20 to about 75, about 25 to about 50, about 10 to about 15, about 15 to about 20, about 20 to about 25, or about 25 to about 30 seconds/period). For example, an oscillation period can be about 10 seconds/period. Oscillation periods can also be asymmetrical, e.g. high substrate concentration for about 2 seconds and low substrate for about 8 seconds, etc. Substrate gradients created by such oscillations drive membrane transporter function and thus drive transport of the substrate.
In some cases, a membrane transporter assay described herein can include providing a cell described herein (e.g., having a membrane transporter and a sensor) with one or more additional agents that help drive membrane transporter function. Examples of additional agents that can help drive membrane transporter function include, without limitation, adenosine triphosphate (ATP), additional ions (e.g., to create an electrochemical gradient).
A membrane transporter assay described herein can include one or more imaging, voltammetric, absorbance or intensitometric methods. In some cases, a membrane transporter assay described herein can include obtaining images of a cell described herein (e.g., having a membrane transporter and a sensor) during perfusion with a substrate solution oscillating between having a high substrate concentration and having a low substrate concentration. For example, at least one instensometric measurement or image can be obtained during perfusion of a cell described herein with a substrate solution having a high substrate concentration and at least one measurement or image can be obtained during perfusion of a cell described herein with a substrate solution having a low substrate concentration. An image can be used to determine the location of a substrate. For example, in cases where a sensor is an intracellular sensor, detection of the substrate indicates that the substrate is intracellular, and absence of detection of the substrate indicates that the substrate is extracellular. Images can be obtained using any appropriate method. In some cases, images can be obtained using a microscope (e.g., confocal microscope). It will be understood that the method of obtaining on image will depend on the type of sensor. For example, in cases where a sensor is a fluorescent sensor, images can be obtained using a fluorescence microscope (e.g., a confocal fluorescence microscope, an epifluorescence microscope, or an inverted fluorescence microscope). In some cases, a microscopic field used to obtain an image can include a view of more than one cell. For example, a microscopic field of view can include a view of at least 2 cells (e.g., at least 5, at least 8, at least 10, at least 12, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 125, at least 150, at least 175, at least 200, or at least 5000 cells). Membrane transporter function in each cell present in a microscopic field of view can be independently measured, permitting large datasets to be collected simultaneously. In some cases, a microscopic field of view including at least 2 cells can include at least 1 experimental cell (e.g., a transporter-positive cell) and at least one control cell (e.g., a transporter-negative cell).
In some cases, a membrane transporter assay described herein can include obtaining a series (e.g., temporal series) of images of a cell having a membrane transporter and a sensor and perfused with a substrate solution oscillating between having a high substrate concentration and having a low substrate concentration. A series of images can be independently obtained or can be frames isolated from a time-lapse movie. Images obtained from a time-lapse movie can be obtained at fast frame rate (e.g., about 2 Hz). A series of images can be obtained manually or automatically. In some cases, a series of images can be stacked, for example, in temporal series. In some cases, a stacked image can provide a readout of the membrane transporter function. For example, a stack of images can provide one or more measurements of membrane transporter function in a time domain. For example, a stack of images can provide one or more measurements of membrane transporter function in a frequency domain. A stack of images described herein can be evaluated using any appropriate image processing software (e.g., ImageJ, FIJI, and ImageJ/FIJI). In some cases, a stack of images described herein can be processed. For example, a stack of images can be subjected to FT analysis.
This document also provides methods of using a membrane transporter assay described herein. In some cases, a membrane transporter assay described herein can be used to characterize and/or analyze membrane transporter function. Any appropriate membrane transporter function can be assayed using a membrane transporter assay described herein. Examples of membrane transporter functions can be assayed include, without limitation, rate of substrate transport, kinetics of substrate transport, and electrophysiology. A method of characterizing membrane transporter function can include expressing a sensor in a cell having a membrane transporter, perfusing the cell with a solution having substrate transported by the membrane transporter, and detecting the substrate. During the perfusion, the substrate solution can oscillate between having a low substrate concentration and having a high substrate concentration. Changes in detection of the substrate can be used to characterize membrane transporter function. In some cases, a change in substrate location can be used to characterize membrane transporter function. A change from detecting intracellular substrate to detecting extracellular substrate can be indicative of substrate transport. A change from detecting extracellular substrate to detecting intracellular substrate can be indicative of substrate transport. In some cases, a change in amount of substrate transported can be used to characterize transport rate. For example, an increase in the amount of substrate transported can be indicative of an increase in transport rate. For example, a decrease in the amount of substrate transported can be indicative of a decrease in transport rate.
Any membrane transporter assay described herein can be a high-throughput assay. A high-throughput membrane transporter assay described herein can be performed in a multi-well cell culture plate. Examples of multi-well cell culture plates that can be used in a high-throughput membrane transporter assay described herein include, without limitation, 6 well plates, 12 well plates, 24 well plates, 48 well plates, 96 well plates, 384 well plates, and 1536 well plates. In some cases, different wells of a multi-well cell culture plate can have cells having the same membrane transporter. For example, different wells of a multi-well cell culture plate having cells having the same membrane transporter can be treated with different substrates (e.g., different candidate substrates). Alternatively, or in parallel, different wells of a multi-well cell culture plate having cells having the same membrane transporter can be treated with the same substrate, but treated with different modulators.
In some cases, OSTA can be used to characterize Cl−/HCO3− exchange. For example, a method of characterizing Cl−/HCO3− exchange can include expressing a fluorescent pH sensor (e.g., a SNARF-5F-AM dye) in a cell having a Cl−/HCO3− exchanger, perfusing the cell with a solution having Cl−, and detecting the Cl−. During the perfusion, the Cl− solution can oscillate between about 8 mM Cl− and having about 158 mM Cl−. Changes in detection of Cl− can be used to characterize Cl−/HCO3− exchange function.
In some cases, OSTA can be used to characterize SO42−/OH− exchange. For example, a method of characterizing SO42−/OH− exchange can include expressing a fluorescent pH sensor (e.g., a SNARF-5F-AM dye) in a cell having a SO42−/OH− exchanger, perfusing the cell with a solution having SO42−, and detecting the SO42−. During the perfusion, the SO42− solution can oscillate between about 0 mM SO42− and about 100 mM SO42−. Changes in detection of SO42− can be used to characterize SO42−/OH− exchange.
In some cases, OSTA can be used to characterize a glucose transporter. For example, a method of characterizing a glucose transporter function can include expressing a fluorescent glucose sensor (e.g., a cytosolic ratiometric peptide) in a cell having a glucose transporter, perfusing the cell with a solution having glucose, and detecting the glucose. During the perfusion, the glucose solution can oscillate between about 0 mM glucose and about 2 mM glucose. Changes in detection of glucose can be used to characterize glucose transporter function.
In some cases, OSTA can be used to characterize a Na+-dependent glucose transporter. For example, a method of characterizing a Na+-dependent glucose transporter function can include expressing a fluorescent glucose sensor (e.g., a cytosolic ratiometric peptide) in a cell having a Na+-dependent glucose transporter, perfusing the cell with a solution having glucose and Na+, and detecting the glucose. During the perfusion, the glucose solution can oscillate between about 0 mM glucose and about 2 mM glucose. Changes in detection of glucose can be used to characterize Na+-dependent glucose transporter function.
In some cases, OSTA can be used to characterize a glutamate transporter. For example, a method of characterizing glutamate transporter function can include expressing a fluorescent glutamate sensor (e.g., iGluSnFR) in a cell having a glutamate transporter, and perfusing the cell with a solution having glutamate (e.g., MSG), and detecting the glutamate. During the perfusion, the glutamate solution can oscillate between about 0 mM glutamate and about 10 mM glutamate. Changes in detection of glutamate can be used to characterize glutamate transporter function.
In some cases, a membrane transporter assay described herein (e.g., OSTA) can be used to identify a substrate for a membrane transporter. A method of identifying a membrane transporter substrate can include expressing a sensor in a cell having a membrane transporter, perfusing the cell with a solution having a candidate substrate, and detecting the candidate substrate. During the perfusion, the substrate solution can oscillate between having a low substrate concentration and having a high substrate concentration. Detection of the candidate substrate can indicate that the candidate substrate is a substrate for the membrane transporter. Failure to detect the candidate substrate can indicate that the candidate substrate is not a substrate for the membrane transporter.
In some cases, a membrane transporter assay described herein can be used to screen for membrane transporter modulators (e.g., agonists or antagonists). For example, OSTA can be used to screen for agonists of membrane transporter function and/or antagonists of membrane transporter function. A membrane transporter modulator can modulate a membrane transporter involved in a disease and/or disorder. A modulator of any appropriate membrane transporter involved in a disease and/or disorder can be assayed using a membrane transporter assay described herein. Examples of diseases and/or disorders associated with membrane transport include, without limitation, cystic fibrosis, cerebral stroke, epilepsy, Alzheimer's disease, Huntington's disease, and amyotrophic lateral sclerosis (ALS). Examples of membrane transporters involved in a disease and/or disorder include, without limitation, SLC26a3, EAAT2, and NBCe1-b. A method of screening for a membrane transporter modulator can include expressing a sensor in a cell having a membrane transporter, perfusing the cell with a solution having substrate transported by the membrane transporter, providing the cell with a candidate modulator, and detecting the substrate. During the perfusion, the substrate solution can oscillate between having a low substrate concentration and having a high substrate concentration. Detection of a change in transport of the substrate (e.g., compared to transport of the substrate in the absence of the candidate modulator) can indicate that the candidate modulator is a modulator for the membrane transporter. For example, an increase in membrane transport function can indicate that the candidate modulator is an agonist for the membrane transporter. For example, a decrease in membrane transport function can indicate that the candidate modulator is an antagonist for the membrane transporter. Detection of the substrate transport can indicate that the candidate modulator is not a modulator for the membrane transporter. In some cases, a modulator of membrane transporter function can be administered to a cell to modulate membrane transporter function. For example, a modulator of membrane transporter function can be administered to a cell to treat (e.g., to reduce the symptoms of) a membrane transporter associated disease or disorder.
The invention will be further described in the following examples, which do not limit the scope of the invention described in the claims.
Current transporter assays rely on single bouts of activity: substrate is added or removed, and resulting changes over time are recorded. In OSTA, however, continuous perfusion provides the ability to add or remove substrate reliably and repeatedly, and the number and rapidity of measurement events is limited only by the combined properties of the experimental setup. OSTA was used to evaluate a wide variety of transporter properties.
A schematic representation of an OSTA is shown in
All experiments presented were performed on HEK-293 (ATCC #CRL-1573) cells transfected using the Amaxa® system (Lonza). Cells were cultured after transfection to densities of 50-90% confluence in 35 mm coverslip-bottomed culture dishes (MatTek), either uncoated or pre-coated with fibronectin (Sigma-Aldrich), and were imaged in situ. Effects of coating on cell adherence were not quantified, but uncoated dishes worked sufficiently well to allow measurements in most cases.
For pH imaging, 50 μg aliquots of SNARF-5F-AM (ThermoFisher S23923) were dissolved in dimethyl sulfoxide (DMSO) to 20 mM, diluted 1:200 (loading concentration 100 μM) in existing culture medium from dish, and cells were incubated for 20-30 minutes under time-lapse imaging (1 frame per 10 seconds) to determine completeness of loading. Cells loaded in this manner provided data for more than 1 hour under constant imaging, although intensities decayed moderately over time. The ratiometric nature of the dye, however, compensated for decay-related artifacts. For glucose imaging, a cytosolic ratiometric genetically encoded glucose sensor (Keller et al, manuscript in preparation) was co-transfected with Sglt2. For glutamate imaging, a version of iGluSnFR (Marvin et al., 2013 Nat. Methods 10:162-170) was used in which the signal peptide and transmembrane domains were removed, thus targeting the sensor to the cytosol.
An off-the-shelf, gravity-fed, four-channel perfusion system (VC3-4, ALA instruments) was used in all experiments. The bottoms of the 60 mL feeder syringes were aligned vertically to 1-2 cm below the microscope stage (outlet level), which provided flow rates of 4-8 mL/minute depending on the fluid level of the syringes. Buffers were gassed by continuous bubbling with 5% CO2 when appropriate. The outlet of the system was directed towards the illuminated area of the coverslip dish at a distance of 3-5 mm, and continuous fast suction at the raised edge of the dish removed solutions. The perfusion outlet was Tygon tubing with 1/16″ inner diameter (somewhat larger than that in the manifold), which might have slowed the velocity of the buffers, allowing for better cell adhesion. Protocols for buffer switching were carried out by the software provided. Buffer changes were characterized to be >90% in 1 second and ˜100% in 1.5 seconds (
All imaging was performed on an LSM-510 (Zeiss) inverted confocal fluorescence microscope, with excitation from argon laser lines at 488 and/or 514 nm, or DPSS at 561 nm. Objectives were Zeiss EC Plan-Neofluar 10×/0.3 NA M27 and Plan-Apochromat 20×/0.8 NA. Separate experiments indicated that conventional epifluorescence microscopes provided similar results. Imaging was carried out at fastest frame rate possible (2 Hz) with bi-directional scanning and pixel dimensions of 512×512 with either 8- or 16-bit image depth. The lower-magnification objective was preferred due to its inclusion of more cells, and although higher magnification always improved signal-to-noise, larger numbers of cells were considered preferable for population sampling. Pinholes were set to their maxima, corresponding to z-depths of 126 μm and 25 μm for the 10× and 20× objectives, respectively, in order to lessen potential motion artifacts in the z dimension. Laser power at 488 and 514 nm was generally set to 100% and nevertheless did not cause noticeable photobleaching, probably because of the fast frame rates, low magnification, and weakness of the aging laser. In the case of the Sglt2 experiment, the DPSS laser was set to 2-10% in the attempt to limit photobleaching of the mRuby2 moiety of the glucose sensor, but even at 2% power photobleaching was unavoidable due to a combination of mRuby2's relative photosensitivity as well as its susceptibility to photobleaching from the 488 nm laser used concurrently. The artifact, while noticeable, did not affect conclusions. Detector gains were always set to prevent overloads of pixels.
SLC26a3 (A3), a Cl−/HCO3− exchanger, is expressed in many tissues where it plays various roles in pH regulation and chloride balance (Ohana et al., 2009 J. Physiol. 587:2179-2185). A3 exchange of Cl−/HCO3− was characterized with the OSTA method (
Another transporter from the SLC26 family, SLC26a2 (A2), has been implicated in a number of osteochondrodysplasias (bone growth abnormalities) due to mutations affecting its normal SO42−/OH− exchange activity (Dawson and Markovich, 2005 Curr. Med. Chem. 12:385-396), which is critical for sulfation of cartilage and extracellular matrix. Normal transport has been reported to require extracellular Cl− to allosterically gate the normal SO42−/OH− exchange function (Ohana et al., 2012 J. Biol. Chem. 287:5122-5132). Thus, an experiment was devised in which permutations of SO42−, Cl−, and OH− could be perfused onto SNARF-5F-loaded cells expressing A2 (
It was noted that the average “tonic” intracellular pH in the absence of extracellular chloride was always lower in A2-expressing cells than in controls. This was consistent with claims that extracellular chloride is required only for influx of OH−, and not efflux (Ohana et al., 2012 J. Biol. Chem. 287:5122-5132). The transport cycle in the absence of Cl− would thus favor efflux of OH− and would lower intracellular pH, as observed herein. The inverse finding, however, was also observed: in the presence of extracellular chloride, tonic intracellular pH was higher in A2-expressing cells than in controls. One possible explanation is that Cl− not only gated OH− entry, but also dictated asymmetry in the transport process, perhaps by allosterically biasing the transporter towards a conformation favorable to OH− influx. In any case, this experiment demonstrates the ease with which transporter function under various permutations of conditions can be quickly yet rigorously tested by the OSTA method.
To explore the OSTA's efficacy on transporters of more diverse types, experiments were designed to test the diabetes-related drug target Sglt2. This Na+/glucose cotransporter is expressed physiologically in the kidney, where, driven by sodium gradients, it recovers the majority of the glucose initially filtered into the urine (Wright, 2001 Am. J. Physiol. Renal Physiol. 280:F10-18). Potent and specific inhibitors have been developed, with the rationale that significant amounts of glucose could be removed from the circulation by blocking recovery and allowing it to be excreted from the body in urine. Some of these inhibitors are clinically approved and in use (Meng et al., 2008 J. Med. Chem. 51:1145-1149). Unfortunately, Sglt2 tends to have a low flux rate in various expression systems (Wright, 2001 Am. J. Physiol. Renal Physiol. 280:F10-18), and is therefore generally difficult to measure in conventional transport assays. To test the measurability of Sglt2's transport under the OSTA paradigm, cells were co-transfected with both Sglt2 and a novel intracellular ratiometric fluorescent glucose sensor protein (Keller et al, manuscript in preparation), and subjected to oscillations in extracellular glucose in the presence of Na+. Because control cells without Sglt2 transfection showed significant background glucose oscillations due to endogenous transport, conditions were optimized to allow for a greater prominence of specific Sglt2 signal. Oscillations of Na+/K+ were superimposed either in conjunction or in opposition to the glucose oscillations, shifting simultaneously both the co-substrate (Na−) gradient as well as the transmembrane potential (K+) driving Sglt2's electrogenic transport (
Due to its prominence and its intricate stoichiometry (3 Na+, 1 H+, and 1 glutamate for 1 K+ (Levy et al., 1998 J. Neurosci. 18:9620-9628)), EAAT2 seemed another appropriate candidate through which to explore further the universality of the method. Excitatory amino acid transporters (EAAT's) were first described in 1978 (Kanner and Sharon, 1978 Biochemistry 17:3949-3953), and have since become well known for their critical role at glutamatergic synapses in the central nervous system (CNS), where they re-uptake the neurotransmitter glutamate and thereby terminate signaling and prevent excitotoxicity (Jensen et al., 2015 Curr. Opin. Pharm. 20:116-123; Nakagawa and Kaneko, 2013 Curr. Mol. Pharmacol. 6:66-73). EAAT2, also known as GLT-1 or SLC1A2, is primarily expressed in astrocytes, and is responsible for the majority of glutamate reuptake in the CNS (Tanaka et al., 1997 Science 276:1699-1702). Defects in EAAT2 are implicated in cerebral stroke, epilepsy, Alzheimer's disease, HIV-associated dementia, Huntington's disease, amyotrophic lateral sclerosis (ALS) and malignant glioma (Shigeri et al., 2004 Brain Res. Rev. 45:250-265), as well as traumatic brain injury (Yi and Hazell, 2006 Neurochem. Int. 48:394-403). EAAT2 is, therefore, a key drug target for these conditions as well as other excitotoxic states.
To evaluate OSTA's efficacy in measuring EAAT-mediated transport, cells were co-transfected with EAAT2 and a cytosolic version of the glutamate sensor iGluSnFR (Marvin et al., 2013 Nat. Methods 10:162-170), and buffers were oscillated between 0 and 10 mM glutamate. This protocol did not produce significant signals. This was likely due to basal levels of intracellular glutamate saturating the sensor, whose Kd is 80 μM (Marvin et al., 2013 Nat. Methods 10:162-170). This sensor-saturation hypothesis was corroborated by depriving EAAT2/iGluSnFR-expressing cells of extracellular glutamate overnight: when glutamate was restored, dF/F rose to a maximum of ˜2.5 (the maximum when recorded on the surface of HEK cells was ˜4-fold (Marvin et al., 2013 Nat. Methods 10:162-170)), remained high in the absence of glutamate, and was unresponsive to subsequent glutamate pulses (
To overcome the obstacles above, cells were exposed to oscillations of extracellular K+ (150 mM, Natfree) of phase opposite that of Na+/glutamate. Through this combined Na+/K+/glutamate stimulus, EAAT2 was apparently induced to run in reverse and export glutamate, lowering intracellular glutamate levels enough to de saturate the sensor and unmask a robust oscillating response of appropriate phase in a subset of iGluSnFR-expressing cells. This signal was then harnessed to test the effects of two compounds. Addition of 2 μM of the EAAT2 inhibitor TFB-TBOA completely abolished the oscillations, with rapid onset and slower washout, consistent with its reported high affinity (IC50˜17 nM (Tsukada et al., 2005 Neuropharmacology 48:479-491)). As a negative control, TFB-TBOA was removed and sodium salicylate (10 mM) was added under the same stimulus, with no effect on EAAT2 activity besides a small tonic baseline decrease, probably due to salicylate's activity as a protonophore (Saeedi et al., 2013 J. Exp. Bot. 64:1829-1836). An equivalent tonic shift was seen in untransfected cells, indicating that it was EAAT2-independent (
Experimental conditions were devised to allow measurement of transport in intracellular organelles. There were two main obstacles to attaining this goal: 1. specific localization of the sensor to the organelle of choice, and 2. rapid control of intracellular substrate concentrations. Glucose transport in the ER was selected since previous work has already shown glucose fluxes occur in the ER (Fehr et al., 2005 Mol. Cell. Biol. 25, 11102-11112), and since an ER-targeted version of the glucose sensor (modified from the sensor used in Example 3; manuscript in preparation) was available in the laboratory. There are numerous well-characterized protein localization motifs, and this therefore represents a general strategy for targeting protein-based sensors to the organelle of choice, and some similar methods exist for dye-based sensors, e.g., targeting pH indicator dyes to organelles (Benink et al., 2009 BioTechniques 47, 769-774). The second issue, manipulation of intracellular substrate concentration (glucose in this case), was overcome by screening six different glucose transporters for their rapidity of glucose transport into the cytosol, as measured using OSTA and the cytosolic version of the glucose sensor (˜3 hours total imaging). It was determined that Glut1 was by far the fastest under the experimental conditions tested, with some cells exhibiting nearly square-wave response. Glut1 expression permeablized the cell membrane to glucose, enabling oscillating glucose stimuli to reach the ER.
To apply OSTA to ER glucose transport, parallel batches of cells were co-transfected with Glut1 and either the cytosolic or the ER-localized sensor, and OSTA was applied under identical conditions (including solutions, imaging, and processing). Responses were large, fast, and saturating for the cytosolic sensor, but were much smaller, slower, and almost completely linear for the ER-localized sensor (
Since OSTA uses constant fast perfusion, temperature-controlled experiments can be carried out using temperature-controlled perfusion alone. To test this, the glucose transport of Glut2, as monitored by the cytosolic version of the same glucose sensor as above, was measured at 26 and 37° C. using OSTA. When the perfusate temperature was changed from 26 to 37° C., an approximately three-fold change was observed in the magnitude of transport rates (
In the analysis stage of the above experiments, finding appropriate ROIs (segmentation) was sometimes difficult. One method was to transfect the transporter of interest fused genetically to a fluorescent reporter protein, but this may have perturbed transporter function and surprisingly did not provide ideal ROIs. This is due to both weak visibility of the reporter due to membrane localization as well as “hot spots” due to non-functional ER-accumulated protein: reporter intensity correlated poorly with functional signal. Another approach taken was to co-transfect a separate cytosolic reporter protein plasmid, but it was observed that cells often seemed to receive only one of the two plasmids, again leading to poor reporter-function correlation. Upon visual inspection of the image stacks, however, time-dependent oscillating functional signatures were visible to the eye. It was realized that these temporal signatures could be computationally identified through Fourier transforms (FTs) of each pixel in the time dimension: pixels with the greatest oscillations would be highlighted in the FT at the stimulus frequency. Since the frequency of the stimulus was known, and the response was presumably of identical frequency, the appropriate images in the Fourier-transformed image stacks (both phase and amplitude) could be selected and used for segmentation of ROIs. This approach is called Fourier Transform OSTA (FT-OSTA).
As a proof-of-principle of this method, data similar to the initial segment of
To benchmark FT-OSTA, substantial artificial Gaussian noise (pixel-wise standard deviations increased ˜10-fold) was added to the same dataset, and results were compared (
To test the method in an actual experimental scenario, a complex and experimentally noisy dataset was re-analyzed using FT-OSTA. Using conventional methods, this dataset appeared to be too noisy to provide definitive information, but by finding the most responsive ROIs through FT-OSTA methods, functional traces were improved enough to allow conclusions to be drawn. The subject of this experiment was the Na+/HCO3− electrogenic co-transporter NBCe1-b, which has a variable stoichiometry of 1 Na+: 2 or 3 HCO3− (Romero et al., 2013 Mol. Aspects Med. 34:159-182). NBCe1-b is critical in renal and ocular physiology, and its mutations have been connected to diseases of these organs (Romero et al., 2013 Mol. Aspects Med. 34:159-182). Since the transporter had previously been found to be electrogenic, altering transmembrane potential by manipulating extracellular K+ concentrations should augment or diminish transport when in conflict or in concert with Na+-driven fluxes. Accordingly, permutations of Na+ and K+ (
In the first stage (I) of the experiment (for this section, refer to
It is to be understood that while the disclosure has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the disclosure, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.
This application claims benefit under 35 U.S.C. § 119(e) to U.S. Application No. 62/562,026 filed on Sep. 22, 2017.
| Number | Date | Country | |
|---|---|---|---|
| 62562026 | Sep 2017 | US |
