PHOTORECEPTOR LIGHT MODULATED CHANNEL--MOLECULAR STUDIES

描述（改编自申请人摘要）：转换

DESCRIPTION (Adapted from applicant's abstract): The conversion of photochemical information into electrical signals takes place in photoreceptor outer segments of the retina. The conductance changes across the plasma membrane are regulated by cGMP-activated ion channels. These channels conduct both sodium and calcium to record transient as well as background changes in light levels. Cyclic nucleotide-gated channels (CNGC) are tetrameric and two subunits, alpha and beta, have been identified. Each subunit has six transmembrane domains and a C-terminal cyclic nucleotide binding domain which is homologous to the cGMP- and cAMP-binding domains of other proteins. There are no structures available for CNGCs, but insights about the channel conformation can be derived from electrophysiological studies of channel function coupled with site-directed mutagenesis in a heterologous expression system. The applicants will explore ligand recognition, investigating specifically the ligand coordination by residues F533, K596 and D604. These residues were predicted to interact with the purine in molecular models based on the coordinates of cAMP bound to the E. coli cAMP-regulatory protein, CRP. The residues will be mutated singly and in combinations to determine their concerted effect on ligand binding. They also will examine the ability of nickel to stabilize the channel opening in the wild type and mutant channels. They will co-express the alpha and beta subunits to determine the influence of the beta subunit on ligand recognition and channel gating. The solvent accessibility of selected residues in the binding domain and their conformational changes upon ligand binding will be investigated using cysteine scanning mutagenesis with methanethiosulfonate reagents. The divalent permeation properties of CNGCs will be examined in order to better understand how the channel regulates the flow of both sodium and calcium ions. Data will be fitted to a two-site model based on Eyring rate theory. The effects of the b subunit on divalent permeation will also be examined. The model can be used to predict the changes in calcium influx as a function of cGMP levels in the cell. The C-terminal cytosolic region of the bovine retina CNGC will be expressed as a soluble protein using a bacterial expression system in order to measure ligand binding to the fragment directly. Limited proteolysis of the binding domain fragment will be used to probe the overall conformation of the apo and ligand-bound forms of the protein. Finally, using highly purified protein fragments, they will crystallize the binding domain in order to determine the X-ray structure of the ligand-protein interaction.