Voltage (V)-operated Ca channels are crucial for signal transduction, and participate in many essential functions. Ca channels (or a closely related class of molecules of skeletal muscle) have an additional function, voltage sensing for excitation-contraction coupling. This duality informs the present proposal, in which knowledge of gating of Ca channels is applied to the skeletal muscle voltage sensor, and, conversely, selected observations on the voltage sensor are extrapolated to expand our understanding of Ca channel gating. Three aspects of gating will be studied in both cardiac (Card) and skeletal (SkM) Ca channels: voltage-dependent inactivation, Ca-dependent inactivation and Ca-mediated positive feedback. There is a Ca2+-binding site that influences each of these functions, probably located on the Ca channel alpha1 subunit. The location and nature of these sites will be explored. First, each site will be characterized functionally in the native system and a mammalian system heterologously expressing """"""""wild type"""""""" channel proteins. Later, the channels will be modified by mutations, and the functional consequences will be evaluated in the expression system. In the cardiac L channel, we will explore the properties of the """"""""priming site"""""""", a Ca-binding site that prevents inactivation of the SkM and Card channels, and then test hypothesis I, that the priming site involves the same regions that determine ion selectivity of permeation. Hypothesis II is that Ca binds to cytoplasmic regions of the alpha1 subunit and causes them to close the channel. Selected regions will be exchanged between cardiac and skeletal alpha1 subunits, or mutated to destroy the Ca-binding character. Hypothesis III is that Ca binding to a cytoplasmic region of alpha1 potentiates activation. Two regions will be mutated to either reduce or increase their Ca-binding ability. To interpret differences in positive feedback between SkM channels from mammals and amphibians, the main subunit of the Ca channel in cardiac and skeletal muscle of Rana Catesbeiana will be cloned. The amphibian clones will then be used in expression experiments analogous to those with the mammalian clones. Results from the work proposed -and from work in other labs- will be interpreted in terms of an evolving model of channel gating, that already incorporates ion diffusional aspects at the inner mouth, and should give a much more detailed account of molecular aspects when this work is done. The present proposal will also be important to advance the understanding of the links between excitation and contraction in muscle.
