DIII-DIV Linker Research Articles

A leaky inactivated state in Nav1.4 created by mutating hydrophobic residues in DIII, DIV S6 segments.

Fast inactivation in Nav channels is essential to shorten the action potential. The canonical model for fast inactivation proposed that the IFM motif in the DIII-DIV linker blocks the channel producing fast inactivation but recent structural studies indicate that large residues in the S6 segments are more likely to act as the fast inactivation gate. Here, we studied the effects of reducing the volume of structurally identified residues in S6 we hypothesize to be part of the inactivation gate. In both DIII and DIV, mutating two large residues to alanine (mutants DIIIAA & DIVAA) creates significant amount of steady state current while none of the single mutations does. In DIII, both individual single mutants significantly shifted the GV curve to the right. However, the GV of the steady current of DIIIAA, showed a shift to the left instead. We hypothesized that by reducing the volumes of the residues in DIII S6, we partially opened the fast inactivation gate, and ions could permeate through the closed inactivated state leading to the apparent left shift of the GV curve. To test this, in the DIIIAA mutant we introduced the IQM mutation, which has been shown to prevent entrance into fast inactivation and as expected, DIIIAA_IQM showed a right-shifted GV curve, similar to the ones seen in single mutations. Finally, we recorded the gating current from DIIIAA and DIVAA, and characterized the charge immobilization. Despite significant steady state current, both DIIIAA and DIVAA showed similar time course and degree of charge immobilization as the wild type. We concluded that by reducing the volumes of the residues, we created a leaky inactivated state in the Nav channels, through which the steady current was conducted. Support NIHR01GM030376.

Hydrophobic residues in S6 segments of nav contribute to the fast inactivation gate

Fast inactivation is essential for physiological function. The sodium current inactivation has been proposed to be a closure of the conduction pathway by the IFM motif between the DIII-DIV linker. However, recent structural data show that the IFM motif is far away from the pore and not likely to block the conduction pathway directly. Searching for a potential inactivation gate, we performed comparative sequence/structure analysis of Navs. We identified two hydrophobic amino acids from each S6 helices that can potentially serve as the gate.

E1784K, the most common Brugada syndrome and long-QT syndrome type 3 mutant, disrupts sodium channel inactivation through two separate mechanisms.

Inheritable and de novo variants in the cardiac voltage-gated sodium channel, Nav1.5, are responsible for both long-QT syndrome type 3 (LQT3) and Brugada syndrome type 1 (BrS1). Interestingly, a subset of Nav1.5 variants can cause both LQT3 and BrS1. Many of these variants are found in channel structures that form the channel fast inactivation machinery, altering the rate, voltage dependence, and completeness of the fast inactivation process. We used a series of mutants at position 1784 to show that the most common inheritable Nav1.5 variant, E1784K, alters fast inactivation through two separable mechanisms: (1) a charge-dependent interaction that increases the noninactivating current characteristic of E1784K; and (2) a hyperpolarized voltage dependence and accelerated rate of fast inactivation that decreases the peak sodium current. Using a homology model built on the NavPaS structure, we find that the charge-dependent interaction is between E1784 and K1493 in the DIII-DIV linker of the channel, five residues downstream of the putative inactivation gate. This interaction can be disrupted by a positive charge at position 1784 and rescued with the K1493E/E1784K double mutant that abolishes the noninactivating current. However, the double mutant does not restore either the voltage dependence or rates of fast inactivation. Conversely, a mutant at the bottom of DIVS4, K1641D, causes a hyperpolarizing shift in the voltage dependence of fast inactivation and accelerates the rate of fast inactivation without causing an increase in noninactivating current. These findings provide novel mechanistic insights into how the most common inheritable arrhythmogenic mixed syndrome variant, E1784K, simultaneously decreases transient sodium currents and increases noninactivating currents, leading to both BrS1 and LQT3.

FGF12A Counteracts Long Qt Syndrome-Linked Inactivation Deficiency

Cardiac sodium channels (Nav1.5) are responsible for the initiation and propagation of the action potential. Multiple proteins interact and modulate its gating kinetics, including FGF12, the most dominant intracellular FGF in human heart. Two isoforms encoded by the FGF12 gene are FGF12a and FGF12b, differing in N-terminal sequences. We investigate the mechanism of FGF12 regulation of Nav1.5 and its interaction with disease-linked mutations. Nav1.5 comprises 4 homologous domains (DI-DIV). Within each domain, 6 transmembrane segments (S1-S6) constitute the voltage sensing domain (VSD; S1-S4) and the channel pore (S5-S6). By probing changes in fluorescence from a fluorophore tethered onto S4, voltage-clamp fluorometry (VCF) allows simultaneous monitoring of individual VSD movement and ion channel kinetics in the cut-open oocyte. Both FGF12 isoforms shifted the steady-state inactivation curve toward depolarizing potentials (FGF12a:+7.4mV, FGF12b:+7.2mV). VCF experiments identified a shift in DI- (FGF12a:+35.5mV, FGF12b:+33.7mV) and DIII-VSD activation (FGF12a:+43.6mV, FGF12b:+34.2mV). The deactivation of DIII-VSD was previously linked to the channel inactivation and the IFM motif within the DIII-DIV linker (Hsu et al, 2017). We hypothesized an IFM role in mediating FGF12 modulation of Nav1.5 kinetics and found that the FGF12 effect on DIII-VSD was removed by the inactivation-deficient IQM(F1486Q) mutant. Interestingly, FGF12a partially rescued the inactivation of IQM, increasing the percentage of inactivated channels at 40mV from 46% relative to the peak to 79%. Consistent results were also found with a long QT Syndrome-linked variant, deltaKPQ, where FGF12a nearly abolished the late Na+ current in this inactivation-deficient mutant. Both FGF12a and FGF12b interact with DIII-DIV linker to modulate NaV1.5 kinetics. FGF12a is able to restore inactivation impaired by disease-linked mutations. A small molecule mimicking the FGF12a effect could be a potent inhibitor of late Na+ current underlying various cardiac pathologies including arrhythmia and heart failure.

Nav1.7 is phosphorylated by Fyn tyrosine kinase which modulates channel expression and gating in a cell type-dependent manner.

Voltage-gated sodium channel Nav1.7 is a key molecule in nociception, and its dysfunction has been associated with various pain disorders. Here, we investigated the regulation of Nav1.7 biophysical properties by Fyn, an Src family tyrosine kinase. Nav1.7 was coexpressed with either constitutively active (FynCA) or dominant negative (FynDN) variants of Fyn kinase. FynCA elevated protein expression and tyrosine phosphorylation of Nav1.7 channels. Site-directed mutagenesis analysis identified two tyrosine residues (Y1470 and Y1471) located within the Nav1.7 DIII-DIV linker (L3) as phosphorylation sites of Fyn. Whole-cell recordings revealed that FynCA evoked larger changes in Nav1.7 biophysical properties when expressed in ND7/23 cells than in Human Embryonic Kidney (HEK) 293 cells, suggesting a cell type-specific modulation of Nav1.7 by Fyn kinase. In HEK 293 cells, substitution of both tyrosine residues with phenylalanine dramatically reduced current amplitude of mutant channels, which was partially rescued by expressing mutant channels in ND7/23 cells. Phenylalanine substitution showed little effect on FynCA-induced changes in Nav1.7 activation and inactivation, suggesting additional modifications in the channel or modulation by interaction with extrinsic factor(s). Our study demonstrates that Nav1.7 is a substrate for Fyn kinase, and the effect of the channel phosphorylation depends on the cell background. Fyn-mediated modulation of Nav1.7 may regulate DRG neuron excitability and contribute to pain perception. Whether this interaction could serve as a target for developing new pain therapeutics requires future study.

Regulation of Na+ channel inactivation by the DIII and DIV voltage-sensing domains.

Functional eukaryotic voltage-gated Na+ (NaV) channels comprise four domains (DI-DIV), each containing six membrane-spanning segments (S1-S6). Voltage sensing is accomplished by the first four membrane-spanning segments (S1-S4), which together form a voltage-sensing domain (VSD). A critical NaV channel gating process, inactivation, has previously been linked to activation of the VSDs in DIII and DIV. Here, we probe this interaction by using voltage-clamp fluorometry to observe VSD kinetics in the presence of mutations at locations that have been shown to impair NaV channel inactivation. These locations include the DIII-DIV linker, the DIII S4-S5 linker, and the DIV S4-S5 linker. Our results show that, within the 10-ms timeframe of fast inactivation, the DIV-VSD is the primary regulator of inactivation. However, after longer 100-ms pulses, the DIII-DIV linker slows DIII-VSD deactivation, and the rate of DIII deactivation correlates strongly with the rate of recovery from inactivation. Our results imply that, over the course of an action potential, DIV-VSDs regulate the onset of fast inactivation while DIII-VSDs determine its recovery.

DIII of Voltage-Gated Na+ Channels Interacts With Inactivation in the Time Domain of Intermediate Inactivation

Background: Dysregulation of human cardiac voltage-gated Na+ channel (hNaV1.5) inactivation predisposes patients to sudden death. hNaV1.5 comprises a requisite α-subunit that contains four domains (DI-DIV); each of these contains a voltage sensing domain (VSD). We have shown that prolonged depolarizing pulses uniquely immobilize the DIII-VSD. However, the significance of this immobilization and its potential connection to inactivation has not been explored. Here, we observe activation of the VSDs in the presence of mutations that disrupt inactivation. Methods: Previously, we created four DNA constructs with a cysteine introduced into the DI-DIV VSDs. Channels encoded by these constructs were expressed in Xenopus oocytes, and cysteine-labeled with TAMRA-MTS fluorophores. Ionic current and fluorescence emission that reflected changes in VSD conformation were simultaneously recorded using the cut-open oocyte configuration. Results: A hydrophobic motif (IFM) on the DIII-DIV linker is essential for inactivation, and we disrupted it with the F1486Q mutation. With F1486Q, activation of the DI-, DII- and DIII-VSDs was unaffected, while DIV-VSD activation was shifted to depolarized potentials (DIV-VSD WT-V1/2=-61.8±3.5 mV, IQM-V1/2=-48.9±3.5 mV, p=0.03), stabilizing its rested stated. Previously, we observed that DIII-VSD deactivation was significantly slowed after prolonged depolarizing pulses (t10-90%=11.6±1.9 ms after 1 ms, 18.6±1.6 ms after 200 ms, p=0.02). In contrast, F1486Q caused faster DIII-VSD deactivation (t10-90%=16.3±2.2 ms after 1 ms, 8.8±0.9 ms after 200 ms, p=0.01), implying the IFM motif also stabilizes the DIII-VSD activated state, but only after prolonged pulses. Conclusions: Our results show a clear interaction of the DIII-VSD with the IFM motif in the 100 ms time domain, which is essential for regulating late Na+ current (INa,L). Our results indicate that INa,L, whose enhancement predisposes patients with heart-failure, diabetes and ischemia to arrhythmia, might be regulated by therapeutically targeting the DIII-VSD.

Nav1.5 C-Terminal Domains Influence Calcium Regulation of Fast Inactivation Separately from Calmodulin Interaction

The cardiac sodium channel (Nav1.5) has a complex ‘intracellular Ca2+ sensing apparatus’ within its C-terminal domain (CTD) consisting of a partial EF-hand domain (CTD-EF) and a calmodulin (CaM) binding IQ motif (CTD-IQ). There are additional CaM binding motifs within the DIII-DIV linker. Variation in intracellular Ca2+ concentration influences the voltage-dependence of steady-state fast inactivation (SSI) by an unclear mechanism. Here, we mutated 16 key residues implicated in the interactions between CTD-EF and CTD-IQ, CTD-IQ and CaM, or DIII-DIV and CaM and explored their impact on NaV1.5 function and biochemistry. We used quantitative yeast-two-hybrid assays to measure effects of mutations on the interaction between the full length CTD on CaM, and evaluated SSI in high (1 µM free Ca2+) and low [Ca2+]i conditions. Using either BAPTA or HEDTA as chelator, 1 µM free [Ca2+]i was sufficient to shift SSI (elicited by 50 ms prepulses) towards depolarized potentials. We observed that 3 of 5 CTD-IQ mutations (F1912A, A1924T and IQ/AA) strongly reduced the interaction between the CTD and CaM. However, these mutations did not affect the [Ca2+]i effect on SSI suggesting that SSI [Ca2+]i sensitivity does not depend on CaM binding to the CTD-IQ. Unexpectedly, single and combination mutations of the CTD-EF (L1786A, F1791A, Q1807A, L1862A, E1788A-D1790A-D1792A-E1799A, E1804A-D1802A) diminished the CTD-CaM interaction, and some of these mutations (L1786A, E1788A-D1790A-D1792A-E1799A, E1804A-D1802A) also suppressed the [Ca2+]i effect on SSI. Indeed, the only mutations we studied that blunted the [Ca2+]i effect on SSI were within the CTD-EF. These results suggest that the CTD-EF influences NaV1.5 [Ca2+]i sensitivity and mutations in this domain can also alter the interaction of the CTD with CaM, but CaM interaction with the CTD-IQ is not required for the effect of intracellular Ca2+ on inactivation.

Calcium-Mediated Reversal of CaM on the Nav 1.2 IQ Motif: Nested Anti-Parallel Sites

Calmodulin (CaM) regulates many signal transduction pathways, binding to both enzymes and intracellular segments of several classes of transmembrane receptors and channels. CaM binds its targets with dissociation constants ranging from pM to µM. CaM regulates the inactivation of the neuronal voltage-gated sodium channel 1.2 (NaV1.2). CaM is considered to be a constitutive subunit: both apo and calcium-saturated C-domain of CaM bind tightly to the IQ motif located in the carboxyl-terminal tail of the channel. Apo CaM binds in a semi-open conformation to the IQ motifs of NaV1.2, 1.5, and 1.6. Calcium-saturated CaM binds to the IQ motif, but also binds to a linker between transmembrane domains III and IV. The calcium-dependent competition for CaM between the DIII-DIV linker and the IQ motif is not well understood. Particularly perplexing is the high affinity of CaM for the IQ motif under both apo and calcium-saturating conditions. Does CaM dissociate to bind the III-IV linker? Could a second CaM bind to the channel? How does CaM change position on the channel after binding calcium? To address these questions, we solved the solution (NMR) structure of calcium-saturated CaM C-domain bound to NaV1.2IQ (2M5E). It reveals that calcium binding triggers a reversal relative to that of bound apo CaM C-domain (2KXW). Several NaV1.2IQ residues participate in the interface of both complexes, but their contacts with CaM are distinct because of differences in the topography of the semi-open and open hydrophobic clefts. Comparison of 2M5E with several other complexes of calcium-saturated CaM C-domain bound to target sequences emphasizes why CaM-binding domains are characterized by so many different sequence “motifs”. Favorable energies of association are determined by noncovalent interactions that are made on an atom-by-atom, rather than residue-by-residue, basis. NIH R01 GM57001

Involvement of Separate Regions of the NAV Channel DIII-IV Linker Revealed through State Dependent Trapping with Genetically Encoded Photochemistry

Fast inactivation in voltage-gated sodium channels results from rapid and reversible conformational changes in the cytoplasmic linker between domains III and IV (DIII-IV) and their putative receptor in the pore region of the channel. Introduced or acquired mutations in this complex lead to defective channel inactivation, yet the precise structural basis for these interactions has not yet been fully described. Using the photocrosslinking (PC) unnatural amino acid p-benzoyl-L-phenyl alanine (pBpa) it is possible to trap close structural interactions (∼3 A) during transient conformational rearrangements thus mapping structural reorganisations associated with different states. The rates at which crosslinking reactions occur can report on the affinity of such interactions. We have incorporated the pBpa in mammalian cells at the putative inactivation peptide (IFMT) motif of the DIII-IV linker and demonstrated state dependent trapping of the transient conformations associated with fast inactivation, with different phenotypes and rates of trapping revealing distinct environmental changes at each site. Incorporation of pBpa at two tyrosine residues, Y1494 and Y1495 of NaV1.5, which have previously been implicated in the coupling of activation and inactivation and to interact with Calmodulin produced channels with normal gating and robust expression. Interestingly, the observed state dependent trapping upon irradiation with UV light during patch clamp experiments was functionally distinct from the state-dependent cross-linking interactions observed within the IFM locus. Thus, trapping at these sites suggests a more extensive involvement of the DIII-DIV linker in fast inactivation gating beyond the canonical IFMT motif.

Resurgent currents turn painfully exciting

Resurgent currents turn painfully exciting

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.

A Double Tyrosine Motif in the Cardiac Sodium Channel Domain III-IV Linker Couples Calcium-dependent Calmodulin Binding to Inactivation Gating

Voltage-gated sodium channels maintain the electrical cadence and stability of neurons and muscle cells by selectively controlling the transmembrane passage of their namesake ion. The degree to which these channels contribute to cellular excitability can be managed therapeutically or fine-tuned by endogenous ligands. Intracellular calcium, for instance, modulates sodium channel inactivation, the process by which sodium conductance is negatively regulated. We explored the molecular basis for this effect by investigating the interaction between the ubiquitous calcium binding protein calmodulin (CaM) and the putative sodium channel inactivation gate composed of the cytosolic linker between homologous channel domains III and IV (DIII-IV). Experiments using isothermal titration calorimetry show that CaM binds to a novel double tyrosine motif in the center of the DIII-IV linker in a calcium-dependent manner, N-terminal to a region previously reported to be a CaM binding site. An alanine scan of aromatic residues in recombinant DIII-DIV linker peptides shows that whereas multiple side chains contribute to CaM binding, two tyrosines (Tyr(1494) and Tyr(1495)) play a crucial role in binding the CaM C-lobe. The functional relevance of these observations was then ascertained through electrophysiological measurement of sodium channel inactivation gating in the presence and absence of calcium. Experiments on patch-clamped transfected tsA201 cells show that only the Y1494A mutation of the five sites tested renders sodium channel steady-state inactivation insensitive to cytosolic calcium. The results demonstrate that calcium-dependent calmodulin binding to the sodium channel inactivation gate double tyrosine motif is required for calcium regulation of the cardiac sodium channel.

Functional Interactions between Distinct Sodium Channel Cytoplasmic Domains through the Action of Calmodulin

Sodium channels are fundamental signaling molecules in excitable cells, and are molecular targets for local anesthetic agents and intracellular free Ca(2+) ([Ca(2+)](i)). Two regions of Na(V)1.5 have been identified previously as [Ca(2+)](i)-sensitive modulators of channel inactivation. These include a C-terminal IQ motif that binds calmodulin (CaM) in different modes depending on Ca(2+) levels, and an immediately adjacent C-terminal EF-hand domain that directly binds Ca(2+). Here we show that a mutation of the IQ domain (A1924T; Brugada Syndrome) that reduces CaM binding stabilizes Na(V)1.5 inactivation, similarly and more extensively than even reducing [Ca(2+)](i). Because the DIII-DIV linker is an essential structure in Na(V)1.5 inactivation, we evaluated this domain for a potential CaM binding interaction. We identified a novel CaM binding site within the linker, validated its interaction with CaM by NMR spectroscopy, and revealed its micromolar affinity by isothermal titration calorimetry. Mutation of three consecutive hydrophobic residues (Phe(1520)-Ile(1521)-Phe(1522)) to alanines in this CaM-binding domain recapitulated the electrophysiology phenotype observed with mutation of the C-terminal IQ domain: Na(V)1.5 inactivation was stabilized; moreover, mutations of either CaM-binding domain abolish the well described stabilization of inactivation by lidocaine. The direct physical interaction of CaM with the C-terminal IQ domain and the DIII-DIV linker, combined with the similarity in phenotypes when CaM-binding sites in either domain are mutated, suggests these cytoplasmic structures could be functionally coupled through the action of CaM. These findings have bearing upon Na(+) channel function in genetically altered channels and under pathophysiologic conditions where [Ca(2+)](i) impacts cardiac conduction.

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.

A novel mutation in SCN5A, delQKP 1507–1509, causing long QT syndrome: : Role of Q1507 residue in sodium channel inactivation

Inherited long QT syndrome (LQTS) is caused by mutations in six genes including SCN5A, encoding the α-subunit of the human cardiac voltage-dependent sodium channel hNa v1.5. In LQT3, various mutations in SCN5A were identified, which produce a gain of channel function. The aim of this study was to screen SCN5A for mutations in a family with the LQT3 phenotype and to analyze the consequences of the mutation on the channel function. By polymerase chain reaction-denaturating high performance liquid chromatography-sequencing, we identified a novel deletion in SCN5A, delQKP 1507–1509, in the DIII–DIV linker of the sodium channel. The hNa v1.5/delQKP1507–1509, hNa v1.5/delQ1507 and hNa v1.5/Q1507A mutants were constructed in vitro, mutant channels were expressed in the tsA201 human cell line and studied using the whole-cell configuration of the patch clamp technique. A persistent inward sodium current of 1–1.5% of maximum currents measured at −30 mV in all mutant sodium channels was recorded, which was nearly completely blocked by the sodium-channel blockers tetrodotoxin and lidocaine. The deletion mutants resulted in a significant shift of steady-state activation to more depolarized voltages. The delQ1507 showed a small shift of steady-state inactivation towards more negative potentials, whereas no significant shifts were observed in both steady-state activation and inactivation in Q1507A compared to the wild-type Na v1.5 sodium channels. The novel SCN5A mutation, delQKP, induces a residual current as previously shown for other SCN5A mutations causing LQTS. DelQKP shares the deletion of Q1507 with the formerly known delKPQ 1505–1507. Our data suggest that Q1507 plays an important role in fast sodium channel inactivation.

Negatively charged residues adjacent to IFM motif in the DIII–DIV linker of hNa V1.4 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 (hNa V1.4). 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.

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

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

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.