This proposal describes an unique approach for a detailed analysis of the activation process of the retinal rod cyclic GMP-gated channel. The light response in retinal photoreceptors causes intracellular levels of cGMP to drop, thereby closing cGMP-gated channels and generating an electrical signal to the brain. Structural and kinetic studies indicate that two different subunits assemble into heteromeric channels, but it is not known how each binding event contributes to activation. A photoaffinity cGMP analog will be used to freeze the channel in different states of activation. Single channel analysis will allow the following questions to be addressed: 1. What are the details of channel activation? This includes identifying the states the channel goes through during activation, and how cooperativity is achieved. 2. How does the P subunit effect activation and at which steps? Results from homomeric channels (formed from subunits) and heteromeric channels will be compared with native channels which express a 240 kD 13 subunit. 3. Do different patterns of subunit occupancy by cGMP lead to different channel behaviors? A detailed analysis of the steps leading to activation of this channel will give insight into how it participates in phototransduction. Cyclic nucleotide-gated channel isoforms have been identified in rods, cones, olfactory neuroepithelium, heart, sperm, and kidney. Knowledge of the contribution of different elements to activation might help to understand the roles these isoforms play in different tissues, and facilitate tissue specific targeting in therapeutic applications.
