A multidisciplinary approach will be used to determine the distribution of ion channels in skeletal muscle ad to study mechanisms for creating, controlling, and maintaining these channel distributions. The density of channels along the length of the cell will be determined with new electrophysiological methods. The site of synthesis of the channels will be determined with in situ hybridization and immunocytochemistry. The enhanced spatial resolution of these electrical and histological techniques is revealing a complex ordering of ion channels. Ion channels are particularly important regulators of excitability in neurons and skeletal muscle. How the control of synthesis and localization of channels and other membrane proteins is accomplished is a fundamental question for all cells. The distribution and single channel properties of voltage-gated sodium channels will be studied with loose patch and tight patch voltage clamp. A new technique which combines both patch voltage clamp and ionophoresis will be used to find the degree of colocalization of sodium channels and acetylcholine receptors. Different distributions of sodium channels in fast and slow muscle may suggest functional roles for these channel distributions. Potassium channel and chloride channel distributions will be studied with the same techniques since there is evidence that these too are nonuniformly distributed in muscle. The pertinence of extending these studies to other channels is that the channels are not regulated in unison either spatially or in response to perturbations. thus it may be possible to identify both general and specific regulatory controls. The control of channel distribution could occur at any of a number of steps. Since skeletal muscle cells are multinucleated, it is possible that nuclei within the same cytoplasm are differentially regulated. This will be studied with in situ hybridization to determine the abundance and location of mRNA for the sodium channel and acetylcholine receptor. Labeled antibodies and snake toxin will be used to follow protein synthesis of these channels. Extracellular and intracellular factors that may regulate channel synthesis, distribution, or immobilization will be tested. These studies will also be done another multinucleated cell, the eel electrocyte, which also segregates membrane proteins (sodium channels, acetylcholine receptors, and sodium-potassium pumps).
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