Closed-state Fast Inactivation Research Articles

Enhanced closed-state inactivation of mutant cardiac sodium channels (SCN5A N1541D and R1632C) through different mechanisms

BackgroundSCN5A variants can be associated with overlapping phenotypes such as Brugada syndrome (BrS), sinus node dysfunction and supraventricular tachyarrhythmias. Our genetic screening of SCN5A in 65 consecutive BrS probands revealed two patients with overlapping phenotypes: one carried an SCN5A R1632C (in domain IV-segment 4), which we have previously reported, the other carried a novel SCN5A N1541D (in domain IV-segment 1). ObjectiveWe sought to reveal whether or not these variants are associated with the same biophysical defects. MethodsWild-type (WT) or mutant SCN5A was expressed in tsA201-cells, and whole-cell sodium currents (hNav1.5/INa) were recorded using patch-clamp techniques. ResultsThe N1541D-INa density, when assessed from a holding potential of -150 mV, was not different from WT-INa as with R1632C-INa, indicating that SCN5A N1541D did not cause trafficking defects. The steady-state inactivation curve of N1541D-INa was markedly shifted to hyperpolarizing potentials in comparison to WT-INa (V1/2-WT: –82.3 ± 0.9 mV, n = 15; N1541D: –108.8 ± 1.6 mV, n = 26, P < .01) as with R1632C-INa. Closed-state inactivation (CSI) was evaluated using prepulses of −90 mV for 1460 ms. Residual N1541D-INa and R1632C-INa were markedly reduced in comparison to WT-INa (WT: 63.8 ± 4.6%, n = 18; N1541D: 15.1 ± 2.3%, n = 19, P < .01 vs WT; R1632C: 5.3 ± 0.5%, n = 15, P < .01 vs WT). Entry into CSI of N1541D-INa was markedly accelerated, and that of R1632C-INa was weakly accelerated in comparison to WT-INa (tau-WT: 65.8 ± 7.4 ms, n = 18; N1541D: 13.7 ± 1.1 ms, n = 19, P < .01 vs WT; R1632C: 39.5 ± 2.9 ms, n = 15, P < .01 vs WT and N1541D). Although N1541D-INa recovered from closed-state fast inactivation at the same rate as WT-INa, R1632C-INa recovered very slowly (tau-WT: 1.90 ± 0.16 ms, n = 10; N1541D: 1.72 ± 0.12 ms, n = 10, P = .41 vs WT; R1632C: 53.0 ± 2.5 ms, n = 14, P < .01 vs WT and N1541D). ConclusionsBoth N1541D-INa and R1632C-INa exhibited marked enhancement of CSI, but through different mechanisms. The data provided a novel understanding of the mechanisms of CSI of INa. Clinically, the enhanced CSI of N1541D-INa leads to a severe loss-of-function of INa at voltages near the physiological resting membrane potential (~–90 mV) of cardiac myocytes; this can be attributable to the patient's phenotypic manifestations.

Role for Fast Inactivation in Domain I of Voltage Gated Sodium Channels

In sodium channels, the voltage sensing S4 segments move outward in response to membrane depolarization to promote activation and fast inactivation of the channel. Domain-specific roles for S4 segments include activation and fast inactivation. In the present work we investigated the role of domain I in activation, deactivation and fast inactivation of the skeletal muscle sodium channel hNaV1.4. Charge reversal at R1 (R219D) produced a significant hyperpolarizing shift of the activation curve, an effect confirmed in an inactivation deficient background (R219D/QQQ) and with gating currents. Charge reversal at residues lower in the S4 segment (R225D, K228D) produced a depolarizing shift of the activation curve. R219D selectively enhanced fast inactivation from closed states (hyperpolarizing shift of the steady-state fast inactivation curve, and accelerated kinetics of closed-state fast inactivation. Fast inactivation from the open state was not affected by R219D. Lesser effects on closed-state fast inactivation were observed for charge reversal at DIS4 residues lower in that voltage sensor. Charge reversal at inner negative countercharges (E171R, S2; D197R, S3) also produced a hyperpolarizing shift of the steady-state fast inactivation curve and accelerated kinetics of closed-state fast inactivation. These results suggest that in the wild type NaV1.4 channel, interactions of inner negative charges with the outer charge in DIS4 restrict outward movement of that voltage sensor in response to weak depolarization, and limit fast inactivation elicited from closed states. This work was supported by NIH 1R15NS093579-01A1 to JRG and NIH P20GM103408 to ISU.

FGF13 modulates the gating properties of the cardiac sodium channel Nav1.5 in an isoform-specific manner

ABSTRACTFGF13 (FHF2), the major fibroblast growth factor homologous factor (FHF) in rodent heart, directly binds to the C-terminus of the main cardiac sodium channel, NaV1.5. Knockdown of FGF13 in cardiomyocytes induces slowed ventricular conduction by altering NaV1.5 function. FGF13 has five splice variants, each of which possess the same core region and C terminus but differing in their respective N termini. Whether and how these alternatively spliced N termini impart isoform-specific regulation of NaV1.5, however, has not been reported. Here, we exploited a heterologous expression to explore the specific modulatory effects of FGF13 splice variants FGF13S, FGF13U and FGF13YV on NaV1.5 function. We found these three splice variants differentially modulated NaV1.5 current density. Although steady-state activation was unaltered by any of the FGF13 isoforms (compared to control cells expressing Nav1.5 but not expressing FGF13), open-state fast inactivation and closed-state fast inactivation were markedly slowed, steady-state availability was significantly shifted toward the depolarizing direction, and the window current was increased by each of FGF13 isoforms. Most strikingly, FGF13S hastened the rate of NaV1.5 entry into the slow inactivation state and induced a dramatic slowing of recovery from inactivation, which caused a large decrease in current after either low or high frequency stimulation. Overall, these data showed the diversity of the roles of the FGF13 N-termini in NaV1.5 channel modulation and suggested the importance of isoform-specific regulation.

Voltage Sensor Domains and Closed-State Inactivation in Sodium Channels

Sodium channels enter into a state of fast inactivation after opening, or directly from closed states. We examined the roles of the four voltage sensor domains in hNaV1.4 in closed-state fast inactivation using a mutagenesis approach. Charge reversing mutations of outer arginine residues in domains I, III and IV depolarized the steady-state fast inactivation curve and accelerated entry. Similar effects on closed-state fast inactivation were observed for charge-reversing mutations of inner negative charges in domains I and IV, suggesting that electrostatic interaction of these residues limits S4 translocation in response to sub-threshold depolarization. This work was supported by NIH 2P20GM103408 to ISU.

Domain III S4 in closed-state fast inactivation: Insights from a periodic paralysis mutation

Heterologous expression of sodium channel mutations in hypokalemic periodic paralysis reveals 2 variants on channel dysfunction. Charge-reducing mutations of voltage sensing S4 arginine residues alter channel gating as typically studied with expression in mammalian cells. These mutations also produce leak currents through the voltage sensor module, as typically studied with expression in Xenopus oocytes. DIIIS4 mutations at R3 in the skeletal muscle sodium channel produce gating defects and omega current consistent with the phenotype of reduced excitability. Here, we confirm DIIIS4 R3C gating defects in the oocyte expression system for fast inactivation and its recovery. We provide novel data for the effects of the cysteine mutation on voltage sensor movement, to further our understanding of sodium channel defects in hypokalemic periodic paralysis. Gating charge movement and its remobilization are selectively altered by the mutation at hyperpolarized membrane potential, as expected with reduced serum potassium.

DIII - DIV Linker Charge Mutations Differentially Affect Open and Closed State Fast Inactivation in Nav1.4

Fast inactivation in sodium channels occurs with channel opening, or in response to depolarization that does not open channels. Inactivation is dependent on the binding of an IFMT motif located in the DIII-DIV linker, flanked by a number of positively charged amino acid residues. We investigated the role of two loci of positive charge on the N terminal side of the IFMT motif, and three loci of charge on the C terminal side. To do this we compared the effects of charge reversing and charge substituting mutations in skeletal muscle sodium channel hNaV1.4. Inactivation from the closed state was prolonged by mutations on the N terminal side, but accelerated by mutations on the C terminal side of the inactivation particle. Open-state fast inactivation was typically prolonged with mutation. Our results suggest that these residues play an important role in the inactivation of sodium channels, and that both charge and structure contribute to these roles. This work was supported by NSF RUI 0235358 to JRG and NIH P20RR16454 to ISU from the INBRE program of the National Center for Research Resources.

Open- and closed-state fast inactivation in sodium 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 hNaV1.4. 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.

Charge Immobilization From The Open And Closed States Of Voltage-Gated Sodium Channels

We compared the immobilization of voltage sensor gating charge during open- and closed-state fast inactivation in skeletal muscle sodium channels. To do this we compared charge movement and its immobilization using ionic and gating current recordings in the cut open oocyte configuration. Charge movement and its immobilization were steeply voltage dependent at subthreshold voltages. Kinetics of charge immobilization during open- or closed-state fast inactivation were more rapid than the concomitant decay of ionic current. The extent of charge immobilized was correlated with the completion of closed-state fast inactivation at the most negative pulse commands, and reached a maximum of 2/3 of the ON gating charge. Anthopleurin-A decreased the votlage dependence of charge immobilization compared to wild type channels. Whereas anthopleurin slowed open-state fast inactivation, the toxin accelerated currrent decrement at voltages for which only closed-state fast inactivation was possible. Anthopleurin selectively accelerated remobilzation of the gating charge in fast inactivated channels without slowing the onset or decreasing the extent of charge immobilized. This work was supported by NIH P20RR016454 to ISU.

Differential effects of paramyotonia congenita mutations F1473S and F1705I on sodium channel gating

We investigated effects of paramyotonia congenita mutations F1473S and F1705I on gating of skeletal muscle Na+ channels. We used on-cell recordings from Xenopus oocytes to compare fast inactivation and deactivation in wild type and mutant channels. Then, we used gating current recordings to determine how these actions of PC mutants might be reflected in their effects on charge movement and its immobilization. F1473S, but not F1705I, accelerated deactivation from the inactivated state and enhanced the remobilization of gating charge. F1473S and F1705I decreased the completion of closed-state fast inactivation, and each mutant decreased charge movement over the voltage range at which channels did not activate. An unexpected result was that F1705I increased the extent of charge immobilization in response to strong depolarization. Our results suggest that the DIV S4-S5 linker mutation F1473S promotes the hyperpolarized position of DIVS4 to accelerate recovery. Inhibition of charge movement by F1473S and F1705I in the absence of channel opening is discussed with respect to their effects on closed-state fast inactivation.

Charge Immobilization of Skeletal Muscle Na + Channels: Role of Residues in the Inactivation Linker

We investigated structural determinants of fast inactivation and deactivation in sodium channels by comparing ionic flux and charge movement in skeletal muscle channels, using mutations of DIII-DIV linker charges. Charge altering and substituting mutations at K-1317, K-1318 depolarized the g( V) curve but hyperpolarized the h ∞ curve. Charge reversal and substitution at this locus reduced the apparent voltage sensitivity of open- and closed-state fast inactivation. These effects were not observed with charge reversal at E-1314, E-1315. Mutations swapping or neutralizing the negative cluster at 1314, 1315 and the positive cluster at 1317, 1318 indicated that local interactions dictate the coupling of activation to fast inactivation. Gating charge was immobilized before channel entry into fast inactivation in hNa V1.4 but to a lesser extent in mutations at K-1317, K-1318. These results suggest that charge is preferentially immobilized in channels inactivating from the open state. Recovery of gating charge proceeded with a single, fast phase in the double mutation K-1317R, K-1318R. This mutation also partially uncoupled recovery from deactivation. Our findings indicate that charged residues near the fast inactivation “particle” allosterically interact with voltage sensors to control aspects of gating in sodium channels.

Negative charges in the DIII-DIV linker of human skeletal muscle Na+ channels regulate deactivation gating.

Charge reversing, neutralizing and substituting mutations at D1309 and EE1314,15 in the DIII-DIV linker of the human skeletal muscle sodium channel hNav1.4 were constructed and expressed in Xenopus oocytes. The effects of these mutations on conductance, inactivation and deactivation were determined using on-cell macropatches. D1309R caused a depolarizing shift of the conductance-voltage (g(V)) curve and increased the apparent valency of activation. D1309R and EE1314,15RR increased time to peak activation. D1309R caused a depolarizing shift of the steady-state fast inactivation curve, whereas EE1314,15RR produced a hyperpolarizing shift and decreased the apparent valency. Charge reversal at either D1309 or EE1314,15 slowed open-state fast inactivation and accelerated closed-state fast inactivation. D1309R accelerated recovery from fast inactivation, whereas EE1314,15RR and EE1314,15QQ slowed recovery. Deactivation from the inactivated state was determined by the delay in the onset to recovery from fast inactivation. Recovery delay was abbreviated for D1309R but was prolonged for EE1314,15RR and EE1314,15QQ. Open-state deactivation was determined from the time constant of the decay (tau D) of tail currents. tau D was slowed by D1309R, D1309E, EE1314,15RR and EE1314,15QQ. Our findings suggest an important role in deactivation gating in hNav1.4 for the negative cluster of charge at EE1314,15. These and previous findings suggest that clusters of negatively and positively charged residues in the hNav1.4 DIII-DIV linker differentially regulate the kinetics of fast inactivation.

Defective fast inactivation recovery and deactivation account for sodium channel myotonia in the I1160V mutant

The skeletal muscle sodium channel mutant I1160V cosegregates with a disease phenotype producing myotonic discharges (observed as muscle stiffness) that are worsened by elevated K+ levels but unaffected by cooling. The I1160V alpha-subunit was co-expressed with the beta1-subunit in Xenopus oocytes. An electrophysiological characterization was undertaken to examine the underlying biophysical characteristics imposed by this mutation. Two abnormalities were found. 1) The voltage dependence of steady-state fast inactivation was reduced in I1160V, which resulted in faster rates of closed-state fast inactivation onset and recovery in I1160V compared with wild-type channels. 2) The rates of deactivation were slower in I1160V than in wild-type channels. Using a computer-simulated model, the combination of both defects elicited myotonic runs under conditions of elevated K+, consistent with the observed phenotype of the mutant.