This project concerns the function of neurotransmitter receptors that directly gate ion channels. Nucleic acid techniques will be used in conjunction with electrophysiological measurements (voltage clamp, patch clamp) to test hypotheses about the role of specific polypeptide domains and of individual amino acids in agonist binding and channel gating. The molecular biological manipulations will employ cDNA clones for the receptors; site-specific mutations of these clones will be constructed. Available enzymatic methods will be used to transcribe the normal and mutated clones. The mRNA will be introduced by microinjection or by membrane fusion into Xenopus oocytes or mammalian cells in culture. The target cells will express the receptors in the cell membrane; the functional ion channels will be studied electrophysiologically. The work will include both the nicotinic acetylcholine receptor of Torpedo electric organ (clones now available and under study) and the receptor from mouse muscle (clones expected soon). Analogous studies will be pursued on the GABA receptor-channel complex of rat and chick brain. Work will continue on assaying fractionated mRNA preparations that express GABA channels and on screening cDNA clones constructed from the active fractions. When sequenced clones are available, voltage-clamp and patch-clamp analysis of specifically constructed mutant channels will begin.
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