amphotericin B ͉ gramicidin ͉ hypokalemic periodic paralysis ͉ membrane potential ͉ muscle weakness

Five hereditary sodium channelopathies of skeletal muscle have been identified. Prominent symptoms are either myotonia or weakness caused by an increase or decrease of muscle fiber excitability. The voltage-gated sodium channel NaV1.4, initiator of the muscle action potential, is mutated in all five disorders. Pathogenetically, both loss and gain of function mutations have been described, the latter being the more frequent mechanism and involving not just the ion-conducting pore, but aberrant pores as well. The type of channel malfunction is decisive for therapy which consists either of exerting a direct effect on the sodium channel, i.e., by blocking the pore, or of restoring skeletal muscle membrane potential to reduce the fraction of inactivated channels.

The role of sodium channel closed-state fast inactivation in membrane excitability is not well understood. We compared open- and closed-state fast inactivation, and the gating charge immobilized during these transitions, in skeletal muscle channel hNa(V)1.4. A significant fraction of total charge movement and its immobilization occurred in the absence of channel opening. Simulated action potentials in skeletal muscle fibers were attenuated when pre-conditioned by sub-threshold depolarization. Anthopleurin A, a site-3 toxin that inhibits gating charge associated with the movement of DIVS4, was used to assess the role of this voltage sensor in closed-state fast inactivation. Anthopleurin elicited opposing effects on the gating mode, kinetics and charge immobilized during open- versus closed-state fast inactivation. This same toxin produced identical effects on recovery of channel availability and remobilization of gating charge, irrespective of route of entry into fast inactivation. Our findings suggest that depolarization promoting entry into fast inactivation from open versus closed states provides access to the IFMT receptor via different rate-limiting conformational translocations of DIVS4.

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Poison dart frogs of the genus Phyllobates secrete lipophilic alkaloid toxins through their skin that were used by Colombian Amerindians to poison the tips of blowdarts. One of the most potent toxins identified is batrachotoxin (BTX) which is an activator of voltage-gated Na þ channels. BTX causes sustained opening of these channels by shifting the voltage-dependent activation to more hyperpolarized potentials and by disabling both fast and slow inactivation. It also alters pore conductance and selectivity. Endogenous Na þ channels of the poison arrow frog have been proposed to be insensitive to lethal amounts of BTX. In this project we aim to identify what confers BTX insensitivity to Na þ channels of the host frog Phyllobates aurotaenia, therefore we cloned its skeletal muscle Na V channel. Total RNA from skeletal muscle of Phyllobates aurotaenia was isolated and cDNA was obtained with degenerate primers. The 1819 amino acids sequence shares 72% sequence identity with the rat Na þ channel Na V 1.4, and 73% with that of the snake Thamnophis sirtalis. The TMs are extremely well conserved (87%) with absolute conservation of S4 in all domains. The N-and C-termini as well as the cytoplasmic linkers between domains are more divergent. The D3-D4 linker containing the IFM motif is highly conserved except for Q1348E and K1350P. The DEKA-motif is also absolutely conserved as are the GGGS gating hinge and the QGFS motifs. BTX is thought to bind in the pore region, from the selectivity filter ring to the pore lining S6 TMs. We have identified two S to A mutations flanking the gating-hinge in domains 1 and 3 that may participate in toxin-insensitivity of the Phyllobates channel by impairing the binding of BTX. Supported by NIH GM68044(AMC) and GM30376(FB) and by COLCIENCIAS1106-12-13836(LF).