The cGMP-activated ion channel of photoreceptors is the fundamental molecular element that generates the electrical response to light in the retina. During phototransduction, the absorption of a photon of light by a rhodopsin molecule results in the reduction of the cytosolic concentration of the second messenger cCMP and the closing of a cGMP- activated channel in the membrane of the photoreceptor outer segment. The closing of this cation selective channel in response to light produces the primary electrical signal that is processed by the visual system. Previous work on the macroscopic light-sensitive current in photoreceptors has indicated that these channels are highly specialized for their role in phototransduction. By cooperatively binding multiple cGMP molecules, these channels behave as rapid molecular switches. The goal of the proposed experiments is to understand the molecular mechanisms that underlie these specializations in the cGMP-activated channel. In particular, the proposal focus on studies of the conformational changes in the channel protein that occur during activation by cGMP. The approach will be to study the opening and closing behavior of single cGMP-activated channels and the alterations in this gating behavior produced by site-specific mutations in the channel protein. The channels will be studied using single-channel patch-clamp recording on cGMP-activated channels expressed from the bovine cDNA clone in Xenopus oocytes or mammalian cultured cell lines. Analysis of the single-channel behavior will assess the number of closed and open conformations of the channel, the allowed conformational transitions, and the relative energy of the various conformations. This analysis will be applied to channels that have their structure altered by site-directed mutagenesis. The interactions between subunits of the channel will be examined by analyzing channels containing both normal and mutant subunits. The nature of the functional alterations that result from defined structural changes will provide valuable insights into the molecular mechanisms of the channel function.
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