Normal resting potential (P1) of myofibers follows the Nernst equation, exhibiting about −85 mV at a normal extracellular K + concentration ([K + ] o ) of 4 mM. Hyperpolarization occurs with decreased [K + ] o , although at [K + ] o < 1.0 mM, myofibers paradoxically depolarize to a second stable potential of −60 mV (P2). In rat myofiber bundles, P2 also was found at more physiological [K + ] o and was associated with inexcitability. To increase the relative frequency of P2 to 50%, [K + ] o needed to be lowered to 1.5 mM. In the presence of the ionophore gramicidin, [K + ] o reduction to only 2.5 mM yielded the same effect. Acetazolamide normalized this increased frequency of P2 fibers. The findings mimic hypokalemic periodic paralysis (HypoPP), a channelopathy characterized by hypokalemia-induced weakness. Of myofibers from 7 HypoPP patients, up to 25% were in P2 at a [K + ] o of 4 mM, in accordance with their permanent weakness, and up to 99% were in P2 at a [K + ] o of 1.5 mM, in accordance with their paralytic attacks. Of 36 HypoPP patients, 25 had permanent weakness and myoplasmic intracellular Na + ([Na + ] i ) overload (up to 24 mM) as shown by in vivo 23 Na-MRI. Acetazolamide normalized [Na + ] i and increased muscle strength. HypoPP myofibers showed a nonselective cation leak of 12–19.5 μS/cm 2 , which may explain the Na + overload. The leak sensitizes myofibers to reduced serum K + , and the resulting membrane depolarization causes the weakness. We postulate that the principle of paradoxical depolarization and loss of function upon [K + ] o reduction may apply to other tissues, such as heart or brain, when they become leaky (e.g., because of ischemia).
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.
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.
We studied the consequences of the Nav1.4 mutation R1448H that is situated in the fourth voltage sensor of the channel and causes paramyotonia, a cold-induced myotonia followed by weakness. Previous work showed that the mutation uncouples inactivation from activation. We measured whole-cell Na(+) currents at 10, 15, 20, and 25°C using HEK293 cells stably transfected with wildtype (WT) and R1448H Na(+) channels. A Markov model was developed the parameters of which reproduced the data measured on WT and R1448H channels in the whole voltage and temperature range. It required an additional transient inactivated state and an additional closed-state inactivation transition not previously described. The model was used to predict single-channel properties, free energy barriers and temperature dependence of rates. It allowed us to draw the following conclusions: i) open-state inactivation results from a two-step process; ii) the channel re-openings that cause paramyotonia originate from enhanced deactivation/reactivation and not from destabilized inactivation; iii) the closed-state inactivation of R1448H is strikingly enhanced. We assume that latter explains the episodic weakness following cold-induced myotonia.
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