Esther Fujimoto

Voltage Sensors in Domains III and IV, but Not I and II, Are Immobilized by Na+ Channel Fast Inactivation

Using site-directed fluorescent labeling, we examined conformational changes in the S4 segment of each domain of the human skeletal muscle sodium channel (hSkM1). The fluorescence signals from S4 segments in domains I and II follow activation and are unaffected as fast inactivation settles. In contrast, the fluorescence signals from S4 segments in domains III and IV show kinetic components during activation and deactivation that correlate with fast inactivation and charge immobilization. These results indicate that in hSkM1, the S4 segments in domains III and IV are responsible for voltage-sensitive conformational changes linked to fast inactivation and are immobilized by fast inactivation, while the S4 segments in domains I and II are unaffected by fast inactivation.

Evolutionary diversification of TTX-resistant sodium channels in a predator–prey interaction

Understanding the molecular genetic basis of adaptations provides incomparable insight into the genetic mechanisms by which evolutionary diversification takes place. Whether the evolution of common traits in different lineages proceeds by similar or unique mutations, and the degree to which phenotypic evolution is controlled by changes in gene regulation as opposed to gene function, are fundamental questions in evolutionary biology that require such an understanding of genetic mechanisms. Here we identify novel changes in the molecular structure of a sodium channel expressed in snake skeletal muscle, tsNaV1.4, that are responsible for differences in tetrodotoxin (TTX) resistance among garter snake populations coevolving with toxic newts. By the functional expression of tsNaV1.4, we show how differences in the amino-acid sequence of the channel affect TTX binding and impart different levels of resistance in four snake populations. These results indicate that the evolution of a physiological trait has occurred through a series of unique functional changes in a gene that is otherwise highly conserved among vertebrates.

A defect in skeletal muscle sodium channel deactivation exacerbates hyperexcitability in human paramyotonia congenita

1 Paramyotonia congenita (PC) is a human hereditary disorder wherein missense mutations in the skeletal muscle sodium channel lead to cold‐exacerbated muscle hyperexcitability. The most common site for PC mutations is the outermost arginine of domain IV segment 4 (human R1448, rat R1441). 2 We examined the rat homologues of two PC mutants with changes at this site: R1441P and R1441C. The R→P mutation leads to the most clinically severe form of the disease. Since PC has so far been attributed to defects in fast inactivation, we expected the R→P substitution to have a more dramatic effect on fast inactivation than R→C. Both mutants (R1441P and R1441C), however, had identical rates and voltage dependence of fast inactivation and activation. 3 R1441P and R1441C also had slowed deactivation, compared with wild‐type, raising the possibility that slowed deactivation, in combination with defective fast inactivation, might be a contributing cause of paramyotonia congenita. Furthermore, deactivation was slower in R1441P than in R1441C, suggesting that the worse phenotype of the human R→P mutation is due to a greater effect on deactivation, and supporting our hypothesis that slowed sodium channel deactivation contributes to paramyotonia congenita. 4 We show that the downstroke of the muscle action potential produced a sodium tail current, and thus slowed deactivation opposes repolarization and therefore leads to hyperexcitability. Hyperexcitability due to slowed deactivation, which has previously been overlooked, also predicts the temperature sensitivity of PC, which has otherwise not been adequately explained.

A Single Residue Differentiates between Human Cardiac and Skeletal Muscle Na+ Channel Slow Inactivation

Slow inactivation determines the availability of voltage-gated sodium channels during prolonged depolarization. Slow inactivation in hNa(V)1.4 channels occurs with a higher probability than hNa(V)1.5 sodium channels; however, the precise molecular mechanism for this difference remains unclear. Using the macropatch technique we show that the DII S5-S6 p-region uniquely confers the probability of slow inactivation from parental hNa(V)1.5 and hNa(V)1.4 channels into chimerical constructs expressed in Xenopus oocytes. Site-directed mutagenesis was used to test whether a specific region within DII S5-S6 controls the probability of slow inactivation. We found that substituting V754 in hNa(V)1.4 with isoleucine from the corresponding position (891) in hNa(V)1.5 produced steady-state slow inactivation statistically indistinguishable from that in wild-type hNa(V)1.5 channels, whereas other mutations have little or no effect on slow inactivation. This result indicates that residues V754 in hNa(V)1.4 and I891in hNa(V)1.5 are unique in determining the probability of slow inactivation characteristic of these isoforms. Exchanging S5-S6 linkers between hNa(V)1.4 and hNa(V)1.5 channels had no consistent effect on the voltage-dependent slow time inactivation constants [tau(V)]. This suggests that the molecular structures regulating rates of entry into and exit from the slow inactivated state are different from those controlling the steady-state probability and reside outside the p-regions.

Differential effects of homologous S4 mutations in human skeletal muscle sodium channels on deactivation gating from open and inactivated states

1 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. 2 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. 3 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. 4 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. 5 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.

Temperature Sensitive Defects in Paramyotonia Congenita Mutants R1448C and T1313M

The biophysical origins of paramyotonia congenita and its exacerbation in cold temperatures were examined. Human skeletal muscle voltage‐gated sodium channels were expressed in Xenopus oocytes and macroscopic currents were recorded from cell‐attached patches. Wild‐type (hNaV1.4) channels were compared to two mutant channel isoforms, T1313M and R1448C. The voltage dependence and temperature sensitivity of activation, fast‐inactivation onset and recovery, and deactivation were studied. Although activation and the onset of fast‐inactivation were temperature sensitive in all three isoforms, and although these properties in mutant channels differed from those in wild‐type channels, they did not account for cold‐exacerbation. Deactivation, however, was disproportionately slower in R1448C, but not in T1313M, than in hNaV1.4. These defects may, at least in part, account for the clinical symptoms of paramyotonia congenita and its exacerbation by cold, and provide a basis for studies into the therapeutic alleviation of these symptoms. Muscle Nerve 30: 277–288, 2004

A novel mechanism associated with idiopathic ventricular fibrillation (IVF) mutations R1232W and T1620M in human cardiac sodium channels

Abstract. Two mutations associated with idiopathic ventricular fibrillation (IVF) are localized within extracellular loops between segments DIIIS1-S2 (R1232W) and DIVS3-S4 (T1620M) of the human cardiac sodium channel (hNaV1.5) α-subunit. We studied wild-type hNaV1.5 channels and hNaV1.5 channels with the R1232W/T1620M double mutation expressed in Xenopus oocytes using the cell-attached macropatch technique. We demonstrate that these mutations destabilize the fast-inactivated state (described with a two-state first-order reaction model) by decreasing reaction valence, accelerating recovery, and slowing the onset of fast inactivation, collectively resulting in delayed decay of macroscopic currents. R1232W/T1620M mutations in hNaV1.5 channels also significantly increase steady-state channel availability, indicating that mutated channels occupy the slow inactivated state less than hNaV1.5 channels. Under the stress of repetitive depolarizing pulses, R1232W/T1620M channels demonstrate less use-dependent current reduction compared to wild-type channels. We propose that increased channel availability coupled with destabilized fast inactivation contributes to the pathological effect of R1232W/T1620M mutations, and leads to increased excitability of cardiac tissue in vivo.

Outer and Central Charged Residues in DIVS4 of Skeletal Muscle Sodium Channels Have Differing Roles in Deactivation

We tested the effects of charge-neutralizing mutations of the eight arginine residues in DIVS4 of the rat skeletal muscle sodium channel (rNa(V)1.4) on deactivation gating from the open and inactivated states. We hypothesized that neutralization of outer or central charges would accelerate the I-to-C transition as measured by recovery delay because these represent a portion of the immobilizable charge. R1Q abbreviated recovery delay as a consequence of reduced charge content. R4Q increased delay, whereas R5Q abbreviated delay, and charge-substitutions at these residues indicated that each effect was allosteric. We also hypothesized that neutralization of any residue in DIVS4 would slow the O-to-C transition with reduction in positive charge. Reduction in charge at R1, and to a lesser extent at R5, slowed open-state deactivation, while charge neutralizations at R2, R3, R4, R6, and R7 accelerated open-state deactivation. Our findings suggest that arginine residues in DIVS4 in rNa(V)1.4 have differing roles in channel closure from open and inactivated states. Furthermore, they suggest that deactivation in DIVS4 is regulated by charge interaction between the electrical field with the outermost residue, and by local allosteric interactions imparted by central charges.

The Delay in Recovery from Fast Inactivation in Skeletal Muscle Sodium Channels Is Deactivation

Abstract1. Using macropatch techniques, we tested the assumption that deactivation underlies the observed delay in the onset to recovery from fast inactivation by comparing open-state deactivation to recovery delay for rat skeletal muscle mutations R1441C and R1441P.2. Deactivation kinetics from the open state were determined from the exponential decay of tail currents. R1441C and R1441P prolonged open-state deactivation, with the greatest effect produced by R1441P.3. Delays in the onset to recovery from fast inactivation for R1441P and for R1441C were abbreviated compared to those for rSkM1. Recovery delay was longer in R1441P than R1441C at voltages more negative than −110 mV. Recovery from inactivation exhibited a voltage dependence which, unlike delay, saturated at depolarized voltages. Recovery rate constants were increased to a similar extent for R1441C and R1441P at −150 to −120 mV compared to rSkMl.4. These results indicate that the delay in the onset to recovery from fast inactivation in skeletal muscle sodium channels is due to deactivation. Lessening of charge immobilization for R1441C and R1441P may contribute to observed biophysical defects underlying the hyperexcitability of muscle fibers containing paramyotonia congenita mutations. The second stage of recovery from fast inactivation may be affected differentially by these mutations.

Negatively charged residues adjacent to IFM motif in the DIII–DIV linker of hNaV1.4 differentially affect slow inactivation

The effects on slow inactivation (SI) of charge substitutions, neutralizations, and reversals were studied for the negatively charged residues D1309 and EE1314,15 surrounding the IFM motif in the DIII–DIV cytoplasmic linker – the putative fast inactivation particle – of human skeletal muscle voltage‐gated sodium channel (hNaV1.4). Changing aspartate (D) at position 1309 to glutamate (E) (substitution) did not strongly affect SI, whereas charge neutralization to glutamine (Q) and charge reversal to arginine (R) right‐shifted the midpoint of the steady‐state SI curve. Charge neutralization (D→Q) at position 1309 also reduced the apparent valence associated with SI. Glutamates (E) at positions 1314 and 1315 were similarly mutated. Charge reversal (EE→RR) right‐shifted the steady‐state SI curve and both reversal and substitution (EE→DD) reduced its apparent valence. Charge neutralization (EE→QQ) and reversal decreased the maximum probability of SI. These mutations also had differential effects on the rate of SI onset and recovery. These results suggest that charged residues in the DIII–DIV linker may interact with structures that control SI.