In skeletal muscle the voltage sensor in the transverse-tubule membranes is thought to control the opening of the Ca2+ release channel in the sarcoplasmic reticulum. Human diseases such as malignant hyperthermia and central core disease may arise from alterations in the activity of the Ca2+ release channel, the voltage sensor, or proteins that modulate the activity of these two proteins. The Ca2+ release channel, also known as the ryanodine receptor, controls the release of Ca2+ from the sarcoplasmic reticulum; an event that triggers the sequence of events that leads to muscle contraction. The voltage sensor, which is also a voltage dependent Ca2+ channel that binds dihydropyridines, appears to directly control the gating of coupled Ca2+ release channels. Other modulatory proteins may regulate the interactions between the voltage sensor and the Ca2+ release channel. Calmodulin, for example, may regulate the conformation of the Ca2+ release channels in a Ca2+ dependent manner. Determining how these proteins regulate each other's activity is the overall goal of the research described in this application. We propose to 1) identify candidate sites on RYR1 for direct binding to the voltage sensor and test the role of these domains in mechanical E-C coupling, 2) evaluate the role of calmodulin in skeletal muscle E-C coupling, and 3) map both the voltage sensor interaction sites in the 3 dimensional reconstruction of RYR1 and the RYR1 interaction sites in a 3D reconstruction of the voltage sensor. Techniques to be used in this application include: protein biochemistry, proteolysis, radioligand binding, IASys biosensor technology, analysis of interactions of GST labeled DHPR fragments with polyHis tagged/Flag labeled fragments of RYR1, the yeast interaction trap system, phage display, site directed mutagenesis, generation of RYR1-RYR2 chimeras, measurement of whole cell Ca2+ currents and Ca2+ transients and cryoelectron microscopy and angular reconstitution.