The proposed research concerns the molecular basis of electrical excitability in nerve, muscle and related cells. The specific focus of our interest is the voltage-regulated sodium channel which mediates the early, regenerative inward currents of the propagated action potential. In our previous work we have isolated the tetrodotoxin (TTX) binding component of the sodium channel from the electric organ of Electrophorus electricus, and have provided information about its size, stability, peptide, amino-acid and carbohydrate compositions as well as preliminary observations of its appearance under the electron microscope, both in solubilized and reconstituted preparations. We have reincorporated this protein into artificial liposomes and demonstrated neurotoxin modulated ion transport. The protein exhibits sites for TTX and STX binding and blockade of transport, and sites for functional binding of alkaloid neurotoxins and local anesthetics, but apparently not for channel specific peptide neurotoxins. Our continuing research represents application of methods of protein biochemistry and membrane biophysics toward elucidation of the protein structure and mechanisms for ion-selective transport, voltage regulation and mechanisms of drug and anesthetic interaction with the channel.
The specific aims i nclude (1) determining the minimal components required to account for channel biophysics and pharmacology, and perfection of methods for their rapid isolation, (2) perfection of biochemical and biophysical assays of channel function, as regulated by voltage or pharmacological reagents, (3) to further describe the structure of the channel by methods of protein chemistry and electron microscopy, (4) to map the protein for sites which may be readily chemically modified, in portions of the molecule involved in conformational changes associated with gating, and (5) where suitable, to apply some of the methods, equipment and technology developed with the sodium channel to problems of other ion channels of excitable membranes.
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