LIPOSOME-PATCH RECORDING OF RECONSTITUTED ION CHANNELS

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The intention of this proposal is to study the relationships between molecular structure and functional behavior of several ion channel proteins from electrically active cells. Three ion channels will be the objects of study: the Ca++-activated K+ channel (CaK) from mammalian muscle and nerve, the voltage-dependent Cl- channel from Torpedo electroplax, and the excitable membrane Na+ channel from mammalian muscle. Single-channel analysis will be applied to all three channels, which will all be studied in model membranes reconstituted from lipids and purified membrane or protein fractions. All experiments proposed are designed to address questions of the underlying molecular mechanisms of observed single-channel behaviors. Specifically, I propose to purify the CaK channel protein in a functionally active form, using a newly discovered scorpion toxin directed against this channel. In addition, the mechanism by which Ca++ activates the channel will be studied, as will modifications of this mechanism in channels from different mammalian sources. Likewise, I will develop an assay, based on Cl- fluxes in liposomes, to detect the presence of the Cl- channel in detergent-solubilized extracts of Torpedo noninnervated-face membranes. The object here will be to carry out the beginning steps for isolating this protein. Finally, the mechanisms of interaction of the Na+ channel with alkaloid toxins, such as veratridine and batrachotoxin, will be studied in planar bilayer membranes.