We propose experiments that will characterize the 200 pS calcium- activated potassium channel of canine colonic smooth muscle. In preliminary work we have examined the characteristics of this channel in cell-attached and excised membrane patches using the patch-clamp technique. Briefly, we find 200 pS channels that are very selective for potassium and show an increased probability of opening in response to elevated intracellular calcium and depolarization. As intracellular calcium is raised over the range of 10-7 to 100-6 M, the voltage range for channel activation shifts more than 100 mV to less depolarized voltages, into the voltage range attained during the contraction cycle. Thus, this channel is likely to mediate a significant repolarizing current under physiological conditions. In this application, we propose a further characterization of the function and regulation of this channel. (1) We will develop the methods necessary for the isolation and reconstitution of this channel into planar lipid bilayers, The reconstituted channels will be characterized in terms of their single channel conductance, calcium- and voltage dependences, and gating kinetics for comparison with the characteristics of the native channels studied in patch-clamp experiments. (2) We will record channel gating in cell-attached patches during superfusion of the cell with calcium ionophores and calcium buffers to study the calcium- dependence of channel function in situ. (3) We will study the effects of apamin, charybdotoxin, and TEA so that this channel may be compared with calcium-activated potassium channels found in other tissues. (4) We will study the effects of the activation of adrenergic and cholinergic receptors on channel gating. These experiments will focus on the modulation on the calcium and voltage- dependence of channel gating. (5) We will investigate the influence o protein phosphorylation and GTP- binding proteins on channel gating. (6) We will compare the properties of channels found in cells isolated from the submucosal and bulk circular layers and the longitudinal layers of the colonic musculature.
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