The purpose of this proposal is to investigate ionic channels in the smooth muscle cell membrane and the control of these channels by membrane potential, neurotransmitters, ions, metabolites and enzymes. These ionic channels control the membrane potential, are the targets of neurotransmission, underly the action potential and are responsible for Ca++ entry into the cell. Consequently, they play a central role in the control of smooth muscle contraction, a topic of considerable therapeutic importance (e.g., in hypertension, vasospasm, and uterine and gastrointestinal contractility). To circumvent the problems associated with studies on tissue, we shall employ a preparation of freshly-dissociated single smooth-muscle cells from Bufo marinus which have been characterized in some detail--electrophysiologically, biochemically and morphologically. The studies will be predominantly electrophysiological with an emphasis on the patch-clamp technology to record currents through single ionic channels and also to record macroscopic currents from the whole cell under conditions where internal cytosolic composition can be altered. There will be five major areas of study: 1) Ca++ channels, 2) Large-conductance Ca++-activated K+ channels, 3) M-current and the action of acetylcholine, 4) The action of certain neuropeptides, and 5) Covalent modification and cytosolic regulators of channels. From these studies, there should emerge a better understanding of the electrical activity of smooth muscle and of those ionic channels common to smooth muscle and other cell types.
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