MOLECULAR MECHANISMS OF GATING IN IONIC CHANNELS

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Biophysical studies are proposed concerning (l) the mechanisms by which excitatory and inhibitory ligand-gated ion channels select between cations and anions, (2) the gate mechanism of a large-conductance K channel at subzero temperatures. The experiments use embryonic rat central neurons (hippocampus, spinal cord) and myotubes maintained in primary tissue culture. The patch clamp technique will be used to study currents from individual channels in excised membrane patches. The excitatory or inhibitory effects of the structurally homologous ion channels operated by the neurotransmitters ACh, GABA, or glycine are determined by the channels' ability to select permeant ions by charge polarity. In the AChR channel, several treatments (high ionic strength, pH, and a carboxyl reagent) will be used to neutralize groups with negative net charges, to assess their importance in the cation/anion selection. In GABA-R and Gly-R channels, the roles of intracellular and extracellular divalent cations in establishing a high selectivity for anions will be studied (a substantial cation leakage through channels exposed to divalent-free salines and partial restoration of anion selectivity by external Ca have observed). The divalent in requirements will be determined and tests will be applied to distinguish between structural and direct electrostatic effects of divalent cations. A new level of kinetic phenomena underlying the gating of large- conductance Ca-activated K channels will be studied at temperatures down to -30 degrees C using techniques developed in our laboratory. These experiments will investigate the operation of the molecular gate by which the ionic current of the channel pore is controlled. At room temperature this gate acts like an instant switch, but at very low temperature channel transitions are slowed to an extent that a cadence of ion current changes can be measured from which the behavior of the actual gate can be inferred. These studies address fundamental questions concerning ionic channels of neurons and muscle, and thus should provide basic information important for understanding normal and impaired function in these tissues.