ISOLATION OF THE LIGHT-MODULATED CHANNEL OF RETINAL RODS

光感受器外部的生化信息的转导

The transduction of biochemical information from the photoreceptor outer segment discs to conductance changes in the plasma membrane is a primary event in visual processing. We now understand that cGMP functions as the terminal, diffusible transmitter in the biochemical cascade to open a plasma membrane ion channel. Multiple cGMP molecules cooperatively bind to the channel to rapidly switch on the membrane conductance. Given this digital-type switch, how does a photoreceptor produce an analog-type response over such a wide range of conditions from total darkness to bright sunlight. Recent experiments by others suggest that Ca plays an important role in adaptation. Our research is focused on answering questions at the molecular level about the nucleotide activation of the channel and the role of Ca in channel function. Our specific aims are 1) to resolve the molecular architecture of the nucleotide binding site on the channel, 2) develop fluorescent or radiolabelled cGMP analogs which can be used to quantitate the cooperative binding isotherm 3) understand the interaction of Ca with the channel and the properties of Na/Ca competition for permeation, 4) to examine possible metabolic regulation of the currents by cAMP, protons, and phosphorylation 5) determine physical dimensions of the channel using a eries of organic cations. Most of the data will be obtained from ionic currents recorded from voltage-clamped plasma membrane excised patches where cGMP or a derivative has been exposed to the cytoplasmic face. Kinetic features of the ligand activation will be obtained from a combination of macroscopic IV recordings, current noise under voltage clamp, instantaneous IV recordings and voltage jumps. The single channel conductance will be estimated from current noise and from individual channel recordings at low concentrations of nucleotide. To determine how divalent cations permeate and block this channel we will use several classical electrophysiological approaches. We can record the current as we change the faction of Na/Ca in the bath. To eliminate gating effects we can also measure the instantaneous IV as we increase the [Ca]. At the present time we view Ca as a permeant channel blocker. What we want to know, in part, is whether the channel selectivity varies with changes in the metabolic state such as phosphorylation or protonation will be done in intact photoreceptors in collaboration with Dr. W. Cobbs. Finally we will probe the physical dimensions of the channel using a series of organic cations beginning with NH4+, a very permeant ion, and increasing the size using TEA, TMA and choline.