Light causes the hyperpolarization of the photoreceptor through a process that is mediated by cGMP and results in the closure of a plasma membrane sodium channel. Elevated intracellular levels of cGMP have been found in certain retinal dystrophies and have been shown to result in photoreceptor degeneration in vitro. The overall objective of this proposal is to investigate the mechanism by which cGMP transfers information from the light stimulus in the disk membrane to the plasma membrane. Recent electrophysiologic investigations have predicted that cGMP is a second messenger in phototransduction. As such, this molecule should have a receptor in the plasma membrane through which it modulates the light response. Previous data have led to the hypothesis that this receptor may be the sodium channel.
The specific aims of this project, therefore, is to define, characterize, quantitate, purify, compare, and subcellularly locate the cGMP receptors in the photoreceptor. To identify and purify the cGMP receptor(s), anti-idiotypic antibodies will be generated to cGMP. These antibodies have been shown in other systems to be invaluable probes to study ligand-receptor interactions. Because these antibodies are produced using the cGMP binding domain of anti-cGMP as the immunogen (the antidody's idiotype), the anti-idiotype should specifically recognize other cGMP binding domains as well. These antibodies can then be used as affinity ligands to purify the receptors, and as immunocytochemical probes to locate the receptors. In addition, the anti-idiotypes often are agonists or antagonists of receptor function and as such can be used to investigate ligand interactions with its receptor. The purified receptors will be examined for function using these antibodies as well as cGMP and its analogs as effectors. The cGMP binding domains will be characterized kinetically and the stoichiometry for its ligand determined. The amino acid sequence of the cGMP binding domains will also be determined and compared to other cyclic nucleotide binding proteins. The results of this project should further our understanding of the mechanism of photo-transduction, the mechanism of photoreceptor degeneration in the retinal dystrophies, and the function of cGMP in other tissues.
