Research Project
3288140
It has recently been observed that guanosine 3':5' cyclic monophosphate ("cGMP") activates cation channels in the outer segments of vertebrate rod photoreceptor cells. Our laboratory has developed a procedure for purification of discs from bovine rod outer segments, and demonstrated that such a channel is associated with discs. When activated by cGMP, the channel can rapidly discharge a pool of Ca++ that is actively accumulated by a disc- associated, ATP-dependent Ca++ pump. Both cGMP and Ca++ are capable of modulating the membrane potential of rod outer segment plasma membranes, and light initiaties events that dramatically alter cGMP metabolism; this suggests that the cGMP-activated channel of discs may be an important link in the short and/or long term regulation of photoreceptor electrical activity. Our goals are to further characterize, reconstitute and purify this cGMP-activated channel. To quantitatively characterize the activation, pharmacology, and conductance properties of the channel, we will employ-- and further refine-- new approaches for rapid kinetic measurement of channel activity. The propsed rapaid kinetic techniques may be of general applicability for characterization of ion channels residing in organelles that are too small for application of electrophysiological methods such as patch clamping. To purify the disc channel, we will employ a novel approach this laboratroy has developed--"transport- specific fractionation"-- together with other methods for membrane protein purification. Transport-specific fractionation uses reconstitution of membrane transport proteins into liposomes as a physical tool for transport protein purification in functionally active form. Identification of the associated agoinst and antagonist binding sites will be an additional means of monitoring the channel's reconstitution and purification. Previously documented regulatory effects of cGMP In other systems appear to be phosphokinases- mediated; in contrast, there are strong indications that both the disc-associated channel-- and a similar or identical one in the plasma membrane of rod other segments-- are directly activated by cGMP. Reconstitution and purification of the channel should enable use to rigorously test this important new hypothesis.
