Cations leaking through the voltage sensor of mutant sodium or calcium channels underlie hypokalaemic periodic paralysis. Groome et al. use muscle fibre recordings, voltage clamp, and molecular dynamics, to investigate recently discovered Nav1.4 channel mutations. They identify a novel voltage sensor movement that may explain the muscle pathology.
In voltage-gated cation channels, a recurrent pattern for mutations is the neutralization of positively charged residues in the voltage-sensing S4 transmembrane segments. These mutations cause dominant ion channelopathies affecting many tissues such as brain, heart, and skeletal muscle. Recent studies suggest that the pathogenesis of associated phenotypes is not limited to alterations in the gating of the ion-conducting alpha pore. Instead, aberrant so-called omega currents, facilitated by the movement of mutated S4 segments, also appear to contribute to symptoms. Surprisingly, these omega currents conduct cations with varying ion selectivity and are activated in either a hyperpolarized or depolarized voltage range. This review gives an overview of voltage sensor channelopathies in general and focuses on pathogenesis of skeletal muscle S4 disorders for which current knowledge is most advanced.
The outermost charged amino acid of S4 segments in the α subunit of human skeletal muscle sodium channels was mutated to cysteine in domains I (R219C), II (R669C), III (K1126C), and IV (R1448C). Double mutations in DIS4 and DIVS4 (R219C/R1448C), DIIS4 and DIVS4 (R669C/R1448C), and DIIIS4 and DIVS4 (K1126C/R1448C) were introduced in other constructs. Macropatch recordings of mutant and wild‐type (hSkM1‐wt) skeletal muscle sodium channels expressed in Xenopus oocytes were used to measure deactivation kinetics from open or fast inactivated states. Conductance (voltage) curves (G (V)) derived from current (voltage) (I (V)) relations indicated a right‐shifted G (V) relationship for R669C and for R669C/R1448C, but not for other mutations. The apparent valency was decreased for all mutations. Time‐to‐peak activation at ‐20 mV was increased for R1448C and for double mutations. Deactivation kinetics from the open state were determined from the monoexponential decay of tail currents. Outermost charge‐to‐cysteine mutations in the S4 segments of domains III and IV slowed deactivation, with the greatest effect produced by R1448C. The deactivation rate constant was slowed to a greater extent for the DIII/DIV double mutation than that calculated from additive effects of single mutations in each of these two domains. Mutation in DIIS4 accelerated deactivation from the open state, whereas mutation in DIS4 had little effect. Delays in the onset to recovery from fast inactivation were determined to assess deactivation kinetics from the inactivated state. Delay times for R219C and R669C were not significantly different from those for hSkM1‐wt. Recovery delay was increased for K1126C, and was accelerated for R1448C. Homologous charge mutations of S4 segments produced domain‐specific effects on deactivation gating from the open and from the fast inactivated state. These results are consistent with the hypothesis that translocations of S4 segments in each domain during deactivation are not identical and independent processes. Non‐identical effects of these mutations raise several possibilities regarding deactivation gating; translocation of DIVS4 may constitute the rate‐limiting step in deactivation from the open state, DIVS4 may be part of the immobilizable charge, and S4 translocations underlying deactivation in human skeletal muscle sodium channel may exhibit co‐operativity.
We studied a two-generation family presenting with conditions that included progressive permanent weakness, myopathic myopathy, exercise-induced contracture before normokalaemic periodic paralysis or, if localized to the tibial anterior muscle group, transient compartment-like syndrome (painful acute oedema with neuronal compression and drop foot). 23Na and 1H magnetic resonance imaging displayed myoplasmic sodium overload, and oedema. We identified a novel familial Cav1.1 calcium channel mutation, R1242G, localized to the third positive charge of the domain IV voltage sensor. Functional expression of R1242G in the muscular dysgenesis mouse cell line GLT revealed a 28% reduced central pore inward current and a −20 mV shift of the steady-state inactivation curve. Both changes may be at least partially explained by an outward omega (gating pore) current at positive potentials. Moreover, this outward omega current of 27.5 nS/nF may cause the reduction of the overshoot by 13 mV and slowing of the upstroke of action potentials by 36% that are associated with muscle hypoexcitability (permanent weakness and myopathic myopathy). In addition to the outward omega current, we identified an inward omega pore current of 95 nS/nF at negative membrane potentials after long depolarizing pulses that shifts the R1242G residue above the omega pore constriction. A simulation reveals that the inward current might depolarize the fibre sufficiently to trigger calcium release in the absence of an action potential and therefore cause an electrically silent depolarization-induced muscle contracture. Additionally, evidence of the inward current can be found in 23Na magnetic resonance imaging-detected sodium accumulation and 1H magnetic resonance imaging-detected oedema. We hypothesize that the episodes are normokalaemic because of depolarization-induced compensatory outward potassium flux through both delayed rectifiers and omega pore. We conclude that the position of the R1242G residue before elicitation of the omega current is decisive for its conductance: if the residue is located below the gating pore as in the resting state then outward currents are observed; if the residue is above the gating pore because of depolarization, as in the inactivated state, then inward currents are observed. This study shows for the first time that functional characterization of omega pore currents is possible using a cultured cell line expressing mutant Cav1.1 channels. Likewise, it is the first calcium channel mutation for complicated normokalaemic periodic paralysis.
1. Serotonin is sequestered by a limited population of identified neurons in the 32 ganglia of the leech nervous system. A major fraction of the serotonin in each ganglion is contained in the paired Retzius cells, colossal effector neurons whose size varies longitudinally. The 5 other classes of identified serotonin-containing neurons, one effector cell and 4 interneurons, are approximately twice as numerous in anterior as in posterior ganglia. 2. We dissected 6 longitudinal samples from the ventral nerve cords of hungry Hirudo medicinalis, and measured their serotonin content using high pressure liquid chromatography with electrochemical detection. A consistent neurochemical pattern emerged in which segmental ganglia 2-4 had the highest quantity of serotonin: 18.51 pmol per ganglion. The anterior cerebral ganglion contained 14.78 pmol, and the content of the 4 posterior samples, segmental ganglia 7-10, 12-15, 17-20 and the caudal ganglion, decreased continuously from 16.35, 15.08, 10.75 to 2.51 pmol per ganglion, respectively. Morphometric analyses indicated that this pattern of ganglionic serotonin correlated primarily with longitudinal variations in the number of serotonin neurons per ganglion and secondarily with volume of the Retzius cells. Retzius cell volume correlated highly with the mass of their innervated body segments both of which are largest in mid-body domains. 3. Serotonin expresses leech feeding, and its ganglionic levels are a potentially useful index of behavioral state. We measured serotonin in the ganglionic samples from hungry and satiated leeches. The samples from recently fed animals contained 28% less serotonin than those from hungry ones. The amounts of serotonin in the cerebral and all the segmental samples from satiated leeches were significantly lower than equivalent samples of hungry animals. A similar pattern of depletion was seen in leeches which fed for a prolonged period (90 to 120 min) rather than the normal period of 30 min. 4. The effects of ingestion on serotonin-containing neurons was examined with the glyoxylic acid-induced histochemical fluorescence. The levels of fluorescence in all serotonin neurons in fed leeches were consistently lower than those in equivalent neurons in hungry animals, corroborating the ganglionic decrease in serotonin in satiated leeches. 5. To examine effects of body wall distension on serotonin levels, hungry leeches were fed to satiation, and half of them were relaxed by removing their distending blood meals. After 6 weeks, ganglionic serotonin in leeches with relaxed bodies was 21% higher than in those with distended bodies. 6. Ingestive behavior depletes serotonin from leech neurons and body wall distension appears to interfere with its synthesis.(ABSTRACT TRUNCATED AT 400 WORDS)
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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