PHYSICAL CHEMISTRY OF NUCLEIC ACIDS

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An interdisciplinary approach, involving molecular biology and electrophysiology, for cloning and studying structure and expression of genes coding for ion channels and neurotransmitter receptors expressed in mammalian brain and skeletal and cardiac muscle is proposed. Major emphasis will be placed on developing a new general method for cloning such genes for those cases in which the conventional approach through protein purification is not readily applicable. The method is based on electrophysiological assays for channels or receptors expressed on the oocyte surface by microinjection of mRNA into Xenopus oocytes, combined with cDNA cloning, and hybrid selection or hybrid arrest procedures. The first goals will be the isolation of genes coding for voltage sensitive calcium channels and for serotonin receptors. The chromosomal genes for the voltage sensitive sodium channels of brain, heart, and muscle will be isolated by standard methods from a presently available partial cDNA clone for the brain gene. Special emphasis will be placed on determining the molecular basis of the diversity of gene products for all the channels of interest, and to the significance of this diversity in signal processing. In all cases, high precision characterization of the electrophysiological properties of the channels expressed in abundance in the oocyte will be carried out by single channel patch clamping methods.