MOLECULAR PHARMACOLOGY OF VOLTAGE-SENSITIVE NA+ CHANNELS

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Voltage-dependent Na-channels are a class of membrane proteins responsible for the rapidly activating and inactivating, inward Na- current that underlies the fast action potential of excitable cells. The long-term objective of this project is to advance understanding of the structure and mechanism of this class of ion channels. The specific aims of this phase of the project focus on three areas of research: (1) Biochemical analysis and investigation of the functional significance of an unusual water- soluble receptor for saxitoxin that is present in bullfrog skeletal muscle. (2) Analysis of the molecular basis for binding of guanidinium toxins such as saxitoxin and tetrodotoxin to a specific receptor site on various Na-channel subtypes. (3) Analysis of the mechanisms of block of Na-channels by diverse inorganic and organic cations, with particular emphasis on high-affinity block of tetrodotoxin-insensitive Na-channels by Zn2+ and on the possibility of multiple sites of action of local anesthetics. Specific binding of (3H) saxitoxin will be used as an assay in the purification of a novel toxin receptor in frog skeletal muscle that may be a water-soluble form of a tetrodotoxin-insensitive Na- channel. Biochemical studies on the purified receptor will be used to establish its structural relationship to known Na-channel proteins. Reconstitution into planar lipid bilayers will be attempted as an approach to the possible Na-channel function of this receptor. Batrachotoxin-activated Na-channels of various subtypes incorporated into planar lipid bilayers will be used as an in vitro model system to study blocking mechanisms at the single channel level. Statistical analysis of toxin blocking events will provide detailed kinetic information on ligand-receptor interactions at the tetrodotoxin binding site. A similar approach will be used to investigate the mechanism of block of Na-channels from canine heart and rat skeletal muscle by Zn2+, local anesthetics, antiarrhythmic and anticonvulsant drugs. An evaluation of the physiological significance of high-affinity Zn2+ block will also be attempted by patch-clamp experiments on cultured cells that express toxin-insensitive Na-channels. The results of this project should further basic understanding of Na-channel biochemistry and pharmacology and may find ultimate applications in musculoskeletal, cardiovascular and neurological diseases.