Voltage-gated calcium channels respond to changes in membrane potential and mediate the entry of calcium into many cell types where it serves as a second messenger to initiate intracellular regulatory and metabolic events. These channels are subject to complex regulation by hormones, neurotransmitters, and second messengers, and are the molecular target for several important classes of drugs. The skeletal muscle calcium channel has two physiological roles. Like calcium channels in other tissues, it mediates voltage-dependent calcium entry into the cytosol. It also serves as a voltage sensor for excitation-contraction coupling linking depolarization of the transverse tubule membrane to release of calcium from the sarcoplasmic reticulum via an unknown mechanism. The abundance of this calcium channel in skeletal muscle have made it a valuable model for studies of the molecular properties of calcium channels. In previous work, we have purified, reconstituted, determined the subunit structure, studied the regulation by protein phosphorylation, and identified 175 kDa and 212 kDa size forms of the principal alpha1 subunit of the skeletal muscle calcium channel. The studies proposed here will combine biochemical, molecular genetic, and electrophysiological approaches to define the functional properties of the individual calcium channel subunits and to establish the structure- function relationships for regulation of calcium channel ion conductance activity by post translational processing and subunit assembly and protein phosphorylation. We will define the carboxyl terminal amino acid sequence of the 175 kDa form of the alpha1 subunit and determine the localization of muscle fibers of its 212 kDa and 175 kDa forms. The ion conductance and regulatory properties of skeletal muscle calcium channels containing the 175 kDa and 212 kDa forms of the alpha1 subunit will be defined. The functional roles of the individual subunits of the calcium channel in expression of its ion conductance activity in mammalian somatic cells will be studied. Physiological sites of phosphorylation of skeletal muscle calcium channels will be identified and their role in regulation of ion conductance activity will be examined. This work will give molecular insight into the complex regulation of the ion conductance activity of this class of calcium channels and equally importantly, will provide a molecular basis for understanding the function and regulation of the L- type calcium channels which are expressed in much lower densities in neurons, cardiac cells.
Showing the most recent 10 out of 77 publications
