The physiological role of the voltage-sensitive sodium channel is mediation of the depolarizing inward currents of the propagated action potential. The electrical organ od Electrophorus electricus is the richest available preparative source of any voltage-sensitive sodium channel. The electroplax protein can be isolated by a one-step immunoaffinity purification that yields a homogeneous preparation with the highest specific activity for tetrodotoxin (TTX) binding yet reported. After reconstitution into liposomes, this protein mediates ion-selective radio-tracer flux when activated by neurotoxins or by chemical modifications which appear to remove inactivation gating. When these liposomes are incorporated into planar bilayers, voltage-dependent single channel activity can be studied in the presence of activating neurotoxins or after chemical modification to remove inactivation gating, in the absence of neurotoxins, when reconstituted liposomes are expanded by a new osmotic fusion protocol, excised patches show a high level of single channel activity, either following treatment with activating neurotoxins, or with voltage alone. In the absence of neurotoxin, two modes of voltage-dependent gating observed. The proposed investigation is aimed at elucidating the molecular structure and mechanism of the electroplax channel. These studies will include mapping the sodium channel for specific sites, including the site of binding of the muscle specific neurotoxin mu-conotoxin, sites of enzymatic phosphorylation by cyclic AMP dependent protein kinase, sites of glycosylation, and domains involved with inactivation gating. (2) In planar bilayer studies, we will complete characterization of the gating, conductance, permeation selectivity and pharmacological properties of sodium channels which have been modified with trypsin of N- bromosuccinimide to remove inactivation gating. The effects of secondary modifications, such as enzymatic phosphorylation, and removal of polysialic acid chains from the protein surface will be studied. (3) With patch-clamped liposomes, we will complete the biophysical description of the rapid kinetics of sodium channels activated with voltage alone, and test the effects of chemical modification. (4) Last, we will pursue the direct structural characterization of the sodium channel by electron microscopy, both in liposomes reconstituted at high density, and in detergent solution, employing specific immunological markers for identification and topographical mapping of the protein. These studies will contribute to our understanding of the molecular structure and mechanism of an important class of ion channel protein.
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