RECONSTITUTION OF CALCIUM CHANNELS IN PLANAR BILAYERS

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The project makes use of planar phospholipid bilayers to study the effects of lipids, ions, and biochemical probes on the conduction and gating characteristics of calcium channels of skeletal muscle transverse tubules. T-tubules are of considerable interest given the great potential that this tissue offers for the purification and molecular cloning of the calcium channel protein. The advantage of planar bilayers for this project is three-fold: it allows recording of unitary calcium currents from channels present in purified t-tubules; it allows experimental control of the bilayer phospholipids surrounding the channel; and it allows control of solutions on both sides of the channel. One specific aim is to understand quantitatively the modulatory role of surface charge on calcium channel conduction and gating kinetics. The interest here is to draw a distinction between charge effects that depend on the lipid polar head group composition and effects controlled by charges present in the channel protein itself. For this purpose, conditions have been found to stabilize the activity of t-tubule calcium channels in two limiting lipid environments: in bilayers composed of the neutral lipid phosphatidylethanolamine or in bilayers of the negatively charged lipid phosphatidylserine. These two lipid compositions will help to define the nature and extent of lipid-protein interactions in calcium channels. A second specific aim is to understand the ways in which Na, Ba, and Ca affect i) how the channel activates and inactivates; ii) the probability of opening vs. voltage curve; and iii) selectivity (Ba, Ca) or loss of selectivity (Na) in the open channel. The interest here is to determine the chemical specificity, lipid sensitivity, and physical location of sites (i.e., close to the gates or within the pore) for current carriers in both, the intracellular and extracellular sides. Mapping of these sites, as well as the location of functional domains within the channel, will be achieved by the use of organic cations as conduction probes, specific calcium channel toxins (atrotoxin, taicatoxin) as probes of gating, and monoclonal antibodies as probes of the subunit composition of functional channels.