Our objective is to characterize single channel potassium currents of axon and muscle membranes. During excitation in nerve and muscle, the initial surge of sodium current is followed by a delayed activation of outward potassium current. Voltage-clamp studies have revealed most, if not all, of the gating, ionic selectivity and pharmacology of potassium currents. Yet, the properties of single potassium channels are virtually unknown. In the case of nerve axons, the resolution of single channels has been particularly elusive. This is largely due to the high density of channels in the axon membrane. Conti and Neher (1980), nevertheless, were able to record single potassium currents from squid axons using patch electrodes. These recordings confirmed earlier predictions derived from noise analyses and revealed new properties of channels which were masked by whole axon currents: the conducting potassium channel is formed by the grouping in time of a collection of short opening events, a process described as channel bursting. We plan to study single potassium channels after reconstitution of purified membrane vesicles into planar phospholipid bilayers. The two sources of membranes chosen for this work, namely lobster axonal membranes from walking leg nerves and ventricular sarcolemma from calf heart, have been extensively purified and characterized. Assembly of bilayers containing membrane fragments will be accomplished by incubating native vesicles with phospholipid monolayers and by apposing two monolayers through an aperture in a film of teflon. A variation of this method which we plan to use, in addition, is to reassemble bilayers on the tip of standard patch-clamp pipettes. This latter system meets the time resolution and current resolution necessary to detect functional potassium channels. The crucial goal of this study is to formulate a molecular picture of potassium channels of nerve and muscle from single channel information. This will be accomplished by a study of reconstituted channels in bilayers composed of simple and defined phopholipids and by focussing into the most relevant gating, ionic selectivity and pharmacological properties.
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