IMMUNO LABELING OF CALCIUM RELEASE CHANNEL PROTEIN

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(Supported by NIH AR40615 to T. Wagenknecht) The main objective of our group is to determine the structure of the ryanodine receptor (RyR), the protein assembly that carries out excitation-contraction coupling in skeletal and heart muscle. The main tools we use are cryo-electron microscopy and image processing. We have labeled a ligand to the receptor with nanogold and, if the binding is successful, the high-resolution EM images of the vitrified specimen should show the RyR with the attached probe at a specific location. This ligand, a peptide toxin, has a direct effect on the open/closed state of the channel. The experiments we are carrying out promise to provide new insights into the mechanism by which stimulation of muscle by nerve leads to contraction, an area that is of intense interest to biomedical scientists because of its implications for understanding and treating muscular, including heart, diseases. We determined the optimal imaging and recording conditions for this work on the IVEM, which will be used to supplement the work done with CTEM. Data collection was started, with good results.