Excitation-contraction coupling (E-C coupling) is a fundamental mechanism responsible for the initiation and control of force in striated muscle. The current hypothesis is that 2 calcium channels are required in this process. 1) Voltage-gated calcium channels, dihydropyridine receptors (DHP-Rs) at T-tubule membranes that act as voltage sensors, and 2) 565-kDa junctional foot proteins (JFPs) or ryanodine receptors that act as calcium release channels on the sarcoplasmic reticulum (SR). Despite intense work on E-C coupling and SR calcium release, the molecular mechanism(s) linking DHP-Rs to JFPs remain unknown. This laboratory has accumulated evidence on a low molecular weight calcium release channel protein in SR that has been overlooked and may provide a key component of E-C coupling. This channel has been studied by a combination of biophysical, biochemical, and immunological techniques and has been named the "sulfhydryl-gated 106-Kda calcium release channel" (SG- 106). This goal of this research is to obtain cDNA clones corresponding to the 106-Kda release channel protein, sequence the complete CDNA and determine the relationship to other, previously characterized channel proteins. Mouse monoclonal and chicken polyclonal antibodies will be raised against the 106-Kda protein isolated from rabbit skeletal muscle. Antibodies will be selected for their reaction with the 106-Kda on Western blots and will be used to isolate pure 106-Kda and screen cDNA libraries from rabbit white skeletal muscle. The immuno-purified 106-kDa protein will be cleaved and the fragments used to obtain partial amino acid sequences. This study will establish whether the 106-kDa protein is a new protein or a fragment of JFPs. Reconstitution of the 106- kDa protein in planar bilayers exhibits the single channel properties of native calcium release channels and thus this protein will be of great interest whether or not it is a fragment of the JFP because it would be readily expressed to study structure- function relationships. %%% Excitation-contraction coupling is a fundamental mechanism responsible for the initiation and control of force in striated muscle. The current hypothesis is that two calcium channels are required for calcium release from an intracellular store in this process. Despite intense work on excitation-contraction coupling the molecular mechanism(s) linking the two calcium channels and how they trigger calcium release remain unknown. This laboratory has obtained evidence that a low molecular weight calcium release channel may also be involved in excitation-contraction coupling. This that has been overlooked by others may provide a key component of the excitation-contraction coupling mechanism. The goal of this research is to isolate and determine the sequence of the gene for this protein. This will provide critical information about its structure and possible relationships to other, known channel proteins and will contribute to understanding of its possible role in excitation-contraction coupling.
