The research proposed seeks to determine how a single population of ion channels transduces the photochemical information of the outer segment into an ionic current that conveys all pre-processed visual information. Gating of the channel is controlled by cooperative binding of multiple cGMP molecules at the cytosolic face of the protein. Previously we examined the structure-function recognition of the ligand by employing a series a cGMP derivatives. We extend these studies using a three- dimensional model of the binding site. With standard molecular modeling approaches, we will dock these derivatives into the binding site and calculate the energies of interaction. Steric constraints will provide an additional test of the model. We will also examine the cooperativity of channel activation at the single channel level. Since channel conductance has been shown to increase stepwise with occupancy of each binding site, we can measure the equilibrium constants for each ligand occupancy directly by measuring the channel opening probability for each current level. Current through these channels is generated by the influx of both Na and Ca ions. Since changes in cytosolic Ca levels are required to adjust the sensitivity of photoreceptors to changes in background light levels, a fundamental question about phototransduction is what fraction of the outer segment current is carried by Ca. The channel could either maintain fixed influx ratios of Na and Ca or alter the ratio in response to varying levels of background light via changes in cytosolic cGMP or Ca levels.
One aim of our research is to determine these influx ratios. Because the ions cannot be distinguished electrophysiologically, we will measure 22Na and 45Ca influx in cultured mammalian cells that transiently express the channels. We showed previously the existence of a high affinity divalent binding site at the cytosolic face of the channel which regulates current at cytosolic divalent levels when cGMP levels are low. Permeation models of the divalent currents suggest that this binding site is outside the permeation path. Using macroscopic and single channel recordings we will explore the possibility that divalent cations allosterically regulate channel function at constant cGMP levels. Site directed mutagenesis will be used to define the channel topology and test the structural models of the binding site.
| Scott, Sean-Patrick; Jarjous, Shadi (2005) Proposed structural mechanism of Escherichia coli cAMP receptor protein cAMP-dependent proteolytic cleavage protection and selective and nonselective DNA binding. Biochemistry 44:8730-48 |
