Changes in the electrical field across the nerve cell membrane are sensed by mobile charged groups associated with the sodium channel macromolecule. In response to a depolarization, the rearrangement of this intramembraneous charge, manifested as 'gating currents', results in a conformational change which opens the channel and allows the flow of ionic current. A major objective of the research proposed here is a more detailed understanding of the link between intramembrane charge movement and the voltage-dependent conformational states of the sodium channel. The sodium channels of squid giant axon and neuroblastoma cells will be studied with a wide range of complementary techniques. Measurements of mean ionic current, admittance, gating currents, ionic and gating current fluctuations, and single Na+ channel currents will provide inter-related details of the conformational changes of the channel macromolecule underlying voltage dependent gating and ion permeation through the open pore. Measurements from large areas of squid axon will be made with an axial wire voltage clamp; the cut-open axon preparation will also be used to study small populations of channels. Neuroblastoma cells will be studied with a combination of patch and whole cell (suction-dialysis) voltage clamp techniques. I have recently made significant improvements in the axial wire voltage clamp resulting in dramatic reductions of background noise levels. These improvements should allow measurements of several phenomena that were previously impossible. Among these are the fluctuations in membrane current resulting from the stochastic movement of intramembraneous charge associated with Na+ channel gating and the Lorentzian noise spectrum resulting from ionic blocking of Na+ channels. Most squid axon experiments will be made in collaboration with Dr. F. Bezanilla at the Marine Biological Laboratory at Woods Hole, Mass. Experiments with neuroblastoma cells, to be conducted in the winter months, are intended to both supplement and be contrasted with results from squid axon. Models of the channel will be developed and tested.
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