ISOLATION OF THE LIGHT-MODULATED CHANNEL OF RETINAL RODS

通过一连串的酶反应，脊椎动物的光感受器

Through a cascade of enzymatic reactions, vertebrate photoreceptors transduce the chemical energy produced by the absorption of a photon by rhodopsin into an electrical signal at the plamsa membrane. The photochemical and biochemical sequences involved in vision are fairly well defined but our understanding of the electrical events on the photoreceptor membrane is rudimentary. This project is designed to provide molecular insight into the rapid changes in photoreceptor outer segment permeability in response to light as well as insight about the membrane structure responsible for these permeability changes. We propose a multidisciplinary approach using familar biochemical and biophysical techniques adapted to the study of this novel transport mechanism. Isolated bovine outer segment membranes, stripped of phosphodiesterase, will be fused into planar lipid bilayers to define the biophysical properties of cyclic GMP gating. Bilayer currents will be compared with cGMP effects on inside-out patches from frog rod outer segments. Electrophysiological techniques will define the cGMP concentration-conductance relationship, single channel properties, ion selectivity, and ion-ion interactions of this novel conductance. Vesicles suitable for 22Na permeability measurements will be formed from ROS membranes in order to assay the cGMP-dependent cation permeability directly. Influx studies will be used to quantitate the 45Ca and 22Na influx under physiological conditions and determine what factors influence the selectivity. Despite the high dissociation constant for cGMP binding to the receptor of 30 uM, 3H-cGMP can be used to define the chemical equilibria of the reaction because of the high site density. The binding cooperativity and receptor density will be estimated. Fluorescence polarization of (thio-fluorescein)-cGMP will be used to determine the binding in solubilized fractions and optimal conditions for detergent solubilization will be defined. Standard biochemical techniques will be used to isolate the cGMP receptor. Purified fractions will be reconstituted into lipid vesicles and assayed for cGMP-dependent permeability changes.