In order to investigate the role in fast inactivation of the cytoplasmic S4‐S5 loop of the fourth domain (IV/S4–S5) within the α‐subunit of the adult human muscle Na+ channel, every single amino acid from R1469 to G1486 was substituted by a cysteine and the mutants were studied by functional expression in human embryonic kidney cells (tsA201) using whole‐cell patch clamping. Effects following intracellular application of the sulfhydryl reagents MTSET and MTSES on the mutants were investigated. Sixteen of eighteen mutants resulted in the formation of functional channels. For P1480C and N1484C, no Na+ currents could be detected in transfected cells. In the absence of sulfhydryl reagents, F1473C and A1481C slowed fast Na+ channel inactivation by 2‐ and 1 .5‐fold, respectively, and L1482C induced a steady‐state Na+ current (Iss) of 3% of peak current (I≥peak) (1 % for wild‐type). Upon application of MTSET and MTSES, changes in fast inactivation gating occurred for most of the mutants. The most dramatic destabilizing effects on fast inactivation were observed for M1476C (9‐fold slowing of inactivation; Iss/Ipeak, 3.6%; + 15 mV shift in steady‐state inactivation; 2‐ to 3‐fold acceleration of recovery from inactivation), A1481C (3‐fold; 14%; +20 mV; no change) and F1473C (2.5‐fold; 2.4%; +8 mV; 1.5‐fold). Less pronounced destabilizing effects were observed for M1477C and L1479C. Strongly stabilizing effects on the inactivated state, that is a 20–30 mV hyperpolarizing shift of the inactivation curve associated with a 3‐ to 4‐fold decrease in the rate of recovery from inactivation, occurred for T1470C, L1471C and A1474C. Almost all effects were independent of the membrane potential; however, A1474C only reacted when cells were depolarized. Significant effects on activation were not observed. We conclude that the IV/S4–S5 loop plays an important role in fast inactivation of the muscle Na+ channel and may contribute to the formation of a receptor for the putative inactivation particle. The effects of sulfhydryl reagents on the various mutations suggest an α‐helical structure of IV/S4–S5 (up to P1480) with destabilizing effects on inactivation for one cluster of amino acids (1473/76/77/79) and a stabilized inactivation at the opposite side of the helix (1470/71/74).
The nematode pharynx has a potassium channel with unusual properties, which allows the muscles to repolarize quickly and with the proper delay. Here, the Caenorhabditis elegans exp-2 gene is shown to encode this channel. EXP-2 is a Kv-type (voltage-activated) potassium channel that has inward-rectifying properties resembling those of the structurally dissimilar human ether-à-go-go-related gene (HERG) channel. Null and gain-of-function mutations affect pharyngeal muscle excitability in ways that are consistent with the electrophysiological behavior of the channel, and thereby demonstrate a direct link between the kinetics of this unusual channel and behavior.
The F1473S mutation of the adult human skeletal muscle Na+ channel causes paramyotonia congenita, a disease characterized by muscle stiffness sometimes followed by weakness in a cold environment. The symptoms are relieved by the local anaesthetic mexiletine. This mutation, which resides in the cytoplasmic S4-S5 loop in domain IV of the alpha-subunit, was studied by heterologous expression in HEK293 cells using standard patch-clamp techniques. Compared to wild-type (WT) channels, those with the F1473S mutation exhibit a twofold slowing of fast inactivation, an increased persistent Na+ current, a +18-mV shift in steady-state inactivation and a fivefold acceleration of recovery from fast inactivation; slow inactivation was similar for both clones. Single-channel recordings for the F1473S mutation revealed a prolonged mean open time and an increased number of channel reopenings that increased further upon cooling. The pharmacological effects of mexiletine on cells expressing either WT, F1473S or G1306E channels were studied. G1306E is a myotonia-causing mutation located within the inactivation gate that displays similar but stronger inactivation defects than F1473S. The hyperpolarizing shift in steady-state inactivation induced by mexiletine was almost identical for all three clones. In contrast, this agent had a reduced effectiveness on the phasic (use-dependent) block of Na+ currents recorded from the mutants: the relative order of block was WT>F1473S>G1306E. We suggest that the relative effectiveness of mexiletine is associated with the degree of abnormal channel inactivation and that the relative binding affinity of mexiletine is not substantially different between the mutations or the WT.
The exp-2 gene in the nematode Caenorhabditis elegans influences the shape and duration of the action potential of pharyngeal muscle cells. Several loss-of-function mutations in exp-2 lead to broadening of the action potential and to a concomitant slowing of the pumping action of the pharynx. In contrast, a gain-of-function mutation leads to narrow action potentials and shallow pumping. We cloned and functionally characterized the exp-2 gene. The exp-2 gene is homologous to genes of the family of voltage-gated K(+) channels (Kv type). The Xenopus oocyte-expressed EXP-2 channel, although structurally closely related to Kv-type channels, is functionally distinct and very similar to the human ether-à-gogo-related gene (HERG) K(+) channel. In response to depolarization, EXP-2 activates slowly and inactivates very rapidly. On repolarization, recovery from inactivation is also rapid and strongly voltage-dependent. These kinetic properties make the Kv-type EXP-2 channel an inward rectifier that resembles the structurally unrelated HERG channel. Apart from many similarities to HERG, however, the molecular mechanism of fast inactivation appears to be different. Moreover, the single-channel conductance is 5- to 10-fold larger than that of HERG and most Kv-type K(+) channels. It appears that the inward rectification mechanism by rapid inactivation has evolved independently in two distinct classes of structurally unrelated, voltage-gated K(+) channels.
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