Membrane permeable local anesthetics modulate NaV1.5 mechanosensitivity

Voltage-gated sodium selective ion channel Na_V1.5 is expressed in the heart and the gastrointestinal tract, which are mechanically active organs. Na_V1.5 is mechanosensitive at stimuli that gate other mechanosensitive ion channels. Local anesthetic and antiarrhythmic drugs act upon Na_V1.5 to modulate activity by multiple mechanisms. This study examined whether Na_V1.5 mechanosensitivity is modulated by local anesthetics. Na_V1.5 channels wereexpressed in HEK-293 cells, and mechanosensitivity was tested in cell-attached and excised inside-out configurations. Using a novel protocol with paired voltage ladders and short pressure pulses, negative patch pressure (-30 mmHg) in both configurations produced a hyperpolarizing shift in the half-point of the voltage-dependence of activation (V_1/2a) and inactivation (V_1/2i) by about -10 mV. Lidocaine (50 µM) inhibited the pressure-induced shift of V_1/2a but not V_1/2i. Lidocaine inhibited the tonic increase in pressure-induced peak current in a use-dependence protocol, but it did not otherwise affect use-dependent block. The local anesthetic benzocaine, which does not show use-dependent block, also effectively blocked a pressure-induced shift in V_1/2a. Lidocaine inhibited mechanosensitivity in Na_V1.5 at the local anesthetic binding site mutated (F1760A). However, a membrane impermeable lidocaine analog QX-314 did not affect mechanosensitivity of F1760A Na_V1.5 when applied from either side of the membrane. These data suggest that the mechanism of lidocaine inhibition of the pressure-induced shift in the half-point of voltage-dependence of activation is separate from the mechanisms of use-dependent block. Modulation of Na_V1.5 mechanosensitivity by the membrane permeable local anesthetics may require hydrophobic access and may involve membrane-protein interactions.     

Membrane permeable local anesthetics modulate NaV1.5 mechanosensitivity

Voltage-gated sodium selective ion channel Na_V1.5 is expressed in the heart and the gastrointestinal tract, which are mechanically active organs. Na_V1.5 is mechanosensitive at stimuli that gate other mechanosensitive ion channels. Local anesthetic and antiarrhythmic drugs act upon Na_V1.5 to modulate activity by multiple mechanisms. This study examined whether Na_V1.5 mechanosensitivity is modulated by local anesthetics. Na_V1.5 channels wereexpressed in HEK-293 cells, and mechanosensitivity was tested in cell-attached and excised inside-out configurations. Using a novel protocol with paired voltage ladders and short pressure pulses, negative patch pressure (-30 mmHg) in both configurations produced a hyperpolarizing shift in the half-point of the voltage-dependence of activation (V_1/2a) and inactivation (V_1/2i) by about -10 mV. Lidocaine (50 µM) inhibited the pressure-induced shift of V_1/2a but not V_1/2i. Lidocaine inhibited the tonic increase in pressure-induced peak current in a use-dependence protocol, but it did not otherwise affect use-dependent block. The local anesthetic benzocaine, which does not show use-dependent block, also effectively blocked a pressure-induced shift in V_1/2a. Lidocaine inhibited mechanosensitivity in Na_V1.5 at the local anesthetic binding site mutated (F1760A). However, a membrane impermeable lidocaine analog QX-314 did not affect mechanosensitivity of F1760A Na_V1.5 when applied from either side of the membrane. These data suggest that the mechanism of lidocaine inhibition of the pressure-induced shift in the half-point of voltage-dependence of activation is separate from the mechanisms of use-dependent block. Modulation of Na_V1.5 mechanosensitivity by the membrane permeable local anesthetics may require hydrophobic access and may involve membrane-protein interactions.     

Ranolazine block of human Nav1.4 sodium channels and paramyotonia congenita mutants

The antianginal drug ranolazine exerts voltage- and use-dependent block (UDB) of several Na⁺ channel isoforms, including Na_v1.4. We hypothesized that ranolazine will similarly inhibit the paramyotonia congenita Na_v1.4 gain-of-function mutations, R1448C, R1448H, and R1448P that are associated with repetitive action potential firing. Whole-cell Na⁺ current (I_Na) was recorded from HEK293 cells expressing the hNa_v1.4 WT or R1448 mutations. At a holding potential (HP) of -140 mV, ranolazine exerted UDB (10 Hz) of WT and R1448 mutations (IC₅₀ = 59 - 71 µM). The potency for ranolazine UDB increased when the frequency of stimulation was raised to 30 Hz (IC₅₀ = 20 - 27 uM). When the HP was changed to -70 mV to mimic the resting potential of an injured skeletal muscle fibre, the potency of ranolazine to block I_Na further increased; values of ranolazine IC₅₀ for block of WT, R1448C, R1448H, and R1448P were 3.8, 0.9, 6.3, and 0.9 uM, respectively. Ranolazine (30 uM) also caused a hyperpolarizing shift in the voltage-dependence of inactivation of WT and R1448 mutations. The effects of ranolazine (30 uM) to reduce I_Na were similar (~35% I_Na inhibition) when different conditioning pulse durations (2-20 msec) were used. Ranolazine (10 µM) suppressed the abnormal I_Na induced by slow voltage ramps for R1448C channels. In computer simulations, 3 µM ranolazine inhibited the sustained and excessive firing of skeletal muscle action potentials that are characteristic of myotonia. Taken together, the data indicate that ranolazine interacts with the open state and stabilizes the inactivated state(s) of Na_v1.4 channels, causes voltage- and use-dependent block of I_Na and suppresses persistent I_Na. These data further suggest that ranolazine might be useful to reduce the sustained action potential firing seen in paramyotonia congenita.     

Block of Tetrodotoxin-sensitive,Nav1.7, and Tetrodotoxin-resistant,Nav1.8, Na+ channels by Ranolazine

Evidence supports a role for the tetrodotoxin-sensitive Nav1.7 and the tetrodotoxin-resistant Nav1.8 in the pathogenesis of pain. Ranolazine, an anti-ischemic drug, has been shown to block cardiac (Nav1.5) late sodium current (I_Na). In this study, whole-cell patch-clamp techniques were used to determine the effects of ranolazine on human Nav1.7 (hNav1.7+β1 subunits) and rat Nav1.8 (rNav1.8) channels expressed in HEK293 and ND7-23 cells, respectively. Ranolazine reduced hNav1.7 and rNav1.8 I_Na with IC₅₀ values of 10.3 and 21.5 μM (holding potential=-120 or -100 mV, respectively). The potency of I_Na block by ranolazine increased to 3.2 and 4.3 μM when 5-sec depolarizing prepulses to -70 (hNav1.7) and -40 (rNav1.8) mV were applied. Ranolazine caused a preferential hyperpolarizing shift of the steady-state fast, intermediate and slow inactivation of hNav1.7 and and intermediate and slow inactivation of rNav1.8, suggesting preferential interaction of the drug with the inactivated states of both channels. Ranolazine (30 μM) caused a use-dependent block (10-msec pulses at 1, 2 and 5 Hz) of hNav1.7 and rNav1.8 I_Na and significantly accelerated the onset of, and slowed the recovery from inactivation, of both channels. An increase of depolarizing pulse duration from 3 to 200 msec did not affect the use-dependent block of I_Na by 100 μM ranolazine. Taken together, the data suggest that ranolazine blocks the open state and may interact with the inactivated states of Nav1.7 and Nav1.8 channels. The state-and use-dependent modulation of hNav1.7 and rNav1.8 Na⁺ channels by ranolazine could lead to an increased effect of the drug at high firing frequencies, as in injured neurons.     

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

FGF13 (FHF2), the major fibroblast growth factor homologous factor (FHF) in rodent heart, directly binds to the C-terminus of the main cardiac sodium channel, Na_V1.5. Knockdown of FGF13 in cardiomyocytes induces slowed ventricular conduction by altering Na_V1.5 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 Na_V1.5, 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 Na_V1.5 function. We found these three splice variants differentially modulated Na_V1.5 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 Na_V1.5 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 Na_V1.5 channel modulation and suggested the importance of isoform-specific regulation.     

Nontruncating SCN1A Mutations Associated with Severe Myoclonic Epilepsy of Infancy Impair Cell Surface Expression

Mutations in SCN1A, encoding the voltage-gated sodium channel Na_V1.1, are the most common cause of severe myoclonic epilepsy of infancy (SMEI) or Dravet syndrome. SMEI is most often associated with premature truncations of Na_V1.1 that cause loss of function, but nontruncating mutations also occur. We hypothesized that some nontruncating mutations might impair trafficking of Na_V1.1 to the plasma membrane. Here we demonstrated that seven nontruncating missense or in-frame deletion mutations (L986F, delF1289, R1648C, F1661S, G1674R, and G1979E) exhibited reduced cell surface expression relative to wild type (WT) Na_V1.1 consistent with impaired trafficking. We tested whether two commonly prescribed antiepileptic drugs (phenytoin, lamotrigine), as well as the cystic fibrosis transmembrane conductance regulator (CFTR) trafficking corrector VRT-325, could rescue cell surface and functional expression of two representative Na_V1.1 mutants (R1648C, G1674R). Treatment of cells with phenytoin increased cell surface expression of WT-Na_V1.1 and both mutant channels, whereas lamotrigine only increased surface expression of R1648C. VRT-325 did not alter surface expression of WT-Na_V1.1 or mutant channels. Although phenytoin increased surface expression of G1674R, channel function was not restored, suggesting that this mutation also causes an intrinsic loss of function. Both phenytoin and lamotrigine increased functional expression of R1648C, but lamotrigine also increased persistent sodium current evoked by this mutation. Our findings indicate that certain nontruncating SCN1A mutations associated with SMEI have impaired cell surface expression and that some alleles may be amenable to pharmacological rescue of this defect. However, rescue of dysfunctional Na_V1.1 channels to the plasma membrane could contribute to exacerbating rather than ameliorating the disease.     

Sensitivity of cloned muscle,heart and neuronal voltage-gated sodium channels to block by polyamines: A possible basis for modulation of excitability in vivo

Spermidine and spermine, are endogenous polyamines (PAs) that regulate cell growth and modulate the activity of numerous ion channel proteins. In particular, intracellular PAs are potent blockers of many different cation channels and are responsible for strong suppression of outward K⁺ current, a phenomenon known as inward rectification characteristic of a major class of K_IR K⁺ channels. We previously described block of heterologously expressed voltage-gated Na⁺ channels (Na_V) of rat muscle by intracellular PAs and PAs have recently been found to modulate excitability of brain neocortical neurons by blocking neuronal Na_V channels. In this study, we compared the sensitivity of four different cloned mammalian Na_V isoforms to PAs to investigate whether PA block is a common feature of Na_V channel pharmacology. We find that outward Na⁺ current of muscle (Na_V1.4), heart (Na_V1.5), and neuronal (Na_V1.2, Na_V1.7) Na_V isoforms is blocked by PAs, suggesting that PA metabolism may be linked to modulation of action potential firing in numerous excitable tissues. Interestingly, the cardiac Na_V1.5 channel is more sensitive to PA block than other isoforms. Our results also indicate that rapid binding of PAs to blocking sites in the Na_V1.4 channel is restricted to access from the cytoplasmic side of the channel, but plasma membrane transport pathways for PA uptake may contribute to long-term Na_V channel modulation. PAs may also play a role in drug interactions since spermine attenuates the use-dependent effect of the lidocaine, a typical local anesthetic and anti-arrhythmic drug.     

Sensitivity of cloned muscle,heart and neuronal voltage-gated sodium channels to block by polyamines

Spermidine and spermine, are endogenous polyamines (PAs) that regulate cell growth and modulate the activity of numerous ion channel proteins. In particular, intracellular PAs are potent blockers of many different cation channels and are responsible for strong suppression of outward K⁺ current, a phenomenon known as inward rectification characteristic of a major class of K_IR K⁺ channels. We previously described block of heterologously expressed voltage-gated Na⁺ channels (Na_V) of rat muscle by intracellular PAs and PAs have recently been found to modulate excitability of brain neocortical neurons by blocking neuronal Na_V channels. In this study, we compared the sensitivity of four different cloned mammalian Na_V isoforms to PAs to investigate whether PA block is a common feature of Na_V channel pharmacology. We find that outward Na⁺ current of muscle (Na_V1.4), heart (Na_V1.5), and neuronal (Na_V1.2, Na_V1.7) Na_V isoforms is blocked by PAs, suggesting that PA metabolism may be linked to modulation of action potential firing in numerous excitable tissues. Interestingly, the cardiac Na_V1.5 channel is more sensitive to PA block than other isoforms. Our results also indicate that rapid binding of PAs to blocking sites in the Na_V1.4 channel is restricted to access from the cytoplasmic side of the channel, but plasma membrane transport pathways for PA uptake may contribute to long-term Na_V channel modulation. PAs may also play a role in drug interactions since spermine attenuates the use-dependent effect of the lidocaine, a typical local anesthetic and anti-arrhythmic drug.     

Comparison of Gating Properties and Use-Dependent Block of Nav1.5 and Nav1.7 Channels by Anti-Arrhythmics Mexiletine and Lidocaine

Mexiletine and lidocaine are widely used class IB anti-arrhythmic drugs that are considered to act by blocking voltage-gated open sodium currents for treatment of ventricular arrhythmias and relief of pain. To gain mechanistic insights into action of anti-arrhythmics, we characterized biophysical properties of Na_v1.5 and Na_v1.7 channels stably expressed in HEK293 cells and compared their use-dependent block in response to mexiletine and lidocaine using whole-cell patch clamp recordings. While the voltage-dependent activation of Na_v1.5 or Na_v1.7 was not affected by mexiletine and lidocaine, the steady-state fast and slow inactivation of Na_v1.5 and Na_v1.7 were significantly shifted to hyperpolarized direction by either mexiletine or lidocaine in dose-dependent manner. Both mexiletine and lidocaine enhanced the slow component of closed-state inactivation, with mexiletine exerting stronger inhibition on either Na_v1.5 or Na_v1.7. The recovery from inactivation of Na_v1.5 or Na_v1.7 was significantly prolonged by mexiletine compared to lidocaine. Furthermore, mexiletine displayed a pronounced and prominent use-dependent inhibition of Na_v1.5 than lidocaine, but not Na_v1.7 channels. Taken together, our findings demonstrate differential responses to blockade by mexiletine and lidocaine that preferentially affect the gating of Na_v1.5, as compared to Na_v1.7; and mexiletine exhibits stronger use-dependent block of Na_v1.5. The differential gating properties of Na_v1.5 and Na_v1.7 in response to mexiletine and lidocaine may help explain the drug effectiveness and advance in new designs of safe and specific sodium channel blockers for treatment of cardiac arrhythmia or pain.     

Block of Brain Sodium Channels by Peptide Mimetics of the Isoleucine,Phenylalanine, and Methionine (IFM) Motif from the Inactivation Gate

Inactivation of sodium channels is thought to be mediated by an inactivation gate formed by the intracellular loop connecting domains III and IV. A hydrophobic motif containing the amino acid sequence isoleucine, phenylalanine, and methionine (IFM) is required for the inactivation process. Peptides containing the IFM motif, when applied to the cytoplasmic side of these channels, produce two types of block: fast block, which resembles the inactivation process, and slow, use-dependent block stimulated by strong depolarizing pulses. Fast block by the peptide ac-KIFMK-NH₂, measured on sodium channels whose inactivation was slowed by the α-scorpion toxin from Leiurus quinquestriatus (LqTx), was reversed with a time constant of 0.9 ms upon repolarization. In contrast, control and LqTx-modified sodium channels were slower to recover from use-dependent block. For fast block, linear peptides of three to six amino acid residues containing the IFM motif and two positive charges were more effective than peptides with one positive charge, whereas uncharged IFM peptides were ineffective. Substitution of the IFM residues in the peptide ac-KIFMK-NH₂ with smaller, less hydrophobic residues prevented fast block. The positively charged tripeptide IFM-NH₂ did not cause appreciable fast block, but the divalent cation IFM-NH(CH₂)₂NH₂ was as effective as the pentapeptide ac-KIFMK-NH₂. The constrained peptide cyclic KIFMK containing two positive charges did not cause fast block. These results indicate that the position of the positive charges is unimportant, but flexibility or conformation of the IFM-containing peptide is important to allow fast block. Slow, use-dependent block was observed with IFM-containing peptides of three to six residues having one or two positive charges, but not with dipeptides or phenylalanine-amide. In contrast to its lack of fast block, cyclic KIFMK was an effective use-dependent blocker. Substitutions of amino acid residues in the tripeptide IFM-NH₂ showed that large hydrophobic residues are preferred in all three positions for slow, use-dependent block. However, substitution of the large hydrophobic residue diphenylalanine or the constrained residues phenylglycine or tetrahydroisoquinoline for phe decreased potency, suggesting that this phe residue must be able to enter a restricted hydrophobic pocket during the binding of IFM peptides. Together, the results on fast block and slow, use-dependent block indicate that IFM peptides form two distinct complexes of different stability and structural specificity with receptor site(s) on the sodium channel. It is proposed that fast block represents binding of these peptides to the inactivation gate receptor, while slow, use-dependent block represents deeper binding of the IFM peptides in the pore.     

Slow Sodium Channel Inactivation and Use-dependent Block Modulated by the Same Domain IV S6 Residue

Voltage- and/or conformation-dependent association and dissociation of local anesthetic-class drugs from a putative receptor site in domain IV S6 of the sodium channel and slow conformation transitions of the drug-associated channel have been proposed as mechanisms of use- and frequency-dependent reduction in sodium current. To distinguish these possibilities, we have explored the reactivity to covalent modification by thiols and block of the mutations F1760C and F1760A at the putative receptor site of the cardiac sodium channel expressed as stable cell lines in HEK-293 cells. Both mutations decreased steady-state fast inactivation, shifting V1/2h from −86 ± 1.3 mV (WT) to −72.3 ± 1.4 mV (F1760C) and −67.7 ± 1 mV (F1760A). In the absence of drug, the F1760C mutant channel displayed use-dependent current reduction during pulse-train stimulation, and faster onset of slow inactivation. This mutant also retained some sensitivity to lidocaine. In contrast, the F1760A mutant showed no use-dependent current reduction or sensitivity to lidocaine. The covalent-modifying agent MTS-ET enhanced use-dependent current reduction of the F1760C mutant channel only. The use-dependent reduction in current of the covalently modified channel completely recovered with rest. Lidocaine produced no additional block during exposure to MTS-ET-treated cells (MTS-ET 43 ± 2.7%: MTS-ET lidocaine 47 ± 4.5%), implying interaction at a common binding site. The data suggest that use-dependent binding at the F1760 site results in enhanced slow inactivation rather than alteration of drug association and dissociation from that site and may be a general mechanism of action of sodium-channel blocking agents.     

Identification of Thr29 as a Critical Phosphorylation Site That Activates the Human Proton Channel Hvcn1 in Leukocytes

Voltage-gated proton channels and NADPH oxidase function cooperatively in phagocytes during the respiratory burst, when reactive oxygen species are produced to kill microbial invaders. Agents that activate NADPH oxidase also enhance proton channel gating profoundly, facilitating its roles in charge compensation and pH_i regulation. The “enhanced gating mode” appears to reflect protein kinase C (PKC) phosphorylation. Here we examine two candidates for PKC-δ phosphorylation sites in the human voltage-gated proton channel, H_V1 (Hvcn1), Thr²⁹ and Ser⁹⁷, both in the intracellular N terminus. Channel phosphorylation was reduced in single mutants S97A or T29A, and further in the double mutant T29A/S97A, by an in vitro kinase assay with PKC-δ. Enhanced gating was evaluated by expressing wild-type (WT) or mutant H_V1 channels in LK35.2 cells, a B cell hybridoma. Stimulation by phorbol myristate acetate enhanced WT channel gating, and this effect was reversed by treatment with the PKC inhibitor GF109203X. The single mutant T29A or double mutant T29A/S97A failed to respond to phorbol myristate acetate or GF109203X. In contrast, the S97A mutant responded like cells transfected with WT H_V1. We conclude that under these conditions, direct phosphorylation of the proton channel molecule at Thr²⁹ is primarily responsible for the enhancement of proton channel gating. This phosphorylation is crucial to activation of the proton conductance during the respiratory burst in phagocytes.     

Sodium Channel Carboxyl-terminal Residue Regulates Fast Inactivation

The Na_v1.2 and Na_v1.3 voltage-gated sodium channel isoforms demonstrate distinct differences in their kinetics and voltage dependence of fast inactivation when expressed in Xenopus oocytes. Co-expression of the auxiliary β1 subunit accelerated inactivation of both the Na_v1.2 and Na_v1.3 isoforms, but it did not eliminate the differences, demonstrating that this property is inherent in the α subunit. By constructing chimeric channels between Na_v1.2 and Na_v1.3, we demonstrate that the carboxyl terminus is responsible for the differences. The Na_v1.2 carboxyl terminus caused faster inactivation in the Na_v1.3 backbone, and the Na_v1.3 carboxyl terminus caused slower inactivation in the Na_v1.2 channel. Through analysis of truncated channels, we identified a homologous 60-amino acid region within the carboxyl terminus of the Na_v1.2 and the Na_v1.3 channels that is responsible for this modulation of fast inactivation. Site-directed replacement of Na_v1.3 lysine 1826 in this region to its Na_v1.2 analogue glutamic acid 1880 (K1826E) shifted the voltage dependence of inactivation toward that of Na_v1.2. The K1826E mutation also accelerated the inactivation kinetics to a level comparable with that of Na_v1.2. The reverse Na_v1.2 E1880K mutation exhibited much slower inactivation kinetics and depolarized inactivation voltage dependence. A complementary mutation located within the inactivation linker of Na_v1.3 (K1453E) caused inactivation changes mirroring those caused by the K1826E mutation in Na_v1.3. Therefore, we have identified a homologous carboxyl-terminal residue that regulates the kinetics and voltage dependence of fast inactivation in sodium channels, possibly via a charge-dependent interaction with the inactivation linker.     

The E1015K Variant in the Synprint Region of the CaV2.1 Channel Alters Channel Function and Is Associated with Different Migraine Phenotypes

Mutations in the CACNA1A gene, which encodes the pore-forming α1A subunit of the Ca_V2.1 voltage-gated calcium channel, cause a number of human neurologic diseases including familial hemiplegic migraine. We have analyzed the functional impact of the E1015K amino acid substitution located in the “synprint” domain of the α1A subunit. This variant was identified in two families with hemiplegic migraine and in one patient with migraine with aura. The wild type (WT) and the E1015K forms of the GFP-tagged α1A subunit were expressed in cultured hippocampal neurons and HEK cells to understand the role of the variant in the transport activity and physiology of Ca_V2.1. The E1015K variant does not alter Ca_V2.1 protein expression, and its transport to the cell surface and synaptic terminals is similar to that observed for WT channels. Electrophysiological data demonstrated that E1015K channels have increased current density and significantly altered inactivation properties compared with WT. Furthermore, the SNARE proteins syntaxin 1A and SNAP-25 were unable to modulate voltage-dependent inactivation of E1015K channels. Overall, our findings describe a genetic variant in the synprint site of the Ca_V2.1 channel which is characterized by a gain-of-function and associated with both hemiplegic migraine and migraine with aura in patients.     

Voltage-sensor movements describe slow inactivation of voltage-gated sodium channels I: Wild-type skeletal muscle NaV1.4

The number of voltage-gated sodium (Na_V) channels available to generate action potentials in muscles and nerves is adjusted over seconds to minutes by prior electrical activity, a process called slow inactivation (SI). The basis for SI is uncertain. Na_V channels have four domains (DI–DIV), each with a voltage sensor that moves in response to depolarizing stimulation over milliseconds to activate the channels. Here, SI of the skeletal muscle channel Na_V1.4 is induced by repetitive stimulation and is studied by recording of sodium currents, gating currents, and changes in the fluorescence of probes on each voltage sensor to assess their movements. The magnitude, voltage dependence, and time course of the onset and recovery of SI are observed to correlate with voltage-sensor movements 10,000-fold slower than those associated with activation. The behavior of each voltage sensor is unique. Development of SI over 1–160 s correlates best with slow immobilization of the sensors in DI and DII; DIII tracks the onset of SI with less fidelity. Showing linkage to the sodium conduction pathway, pore block by tetrodotoxin affects both SI and immobilization of all the sensors, with DI and DII significantly suppressed. Recovery from SI correlates best with slow restoration of mobility of the sensor in DIII. The findings suggest that voltage-sensor movements determine SI and thereby mediate Na_V channel availability.     

Electrophysiological Characteristics of a SCN5A Voltage Sensors Mutation R1629Q Associated With Brugada Syndrome

Brugada syndrome (BrS) is an inherited arrhythmogenic syndrome leading to sudden cardiac death, partially associated with autosomal dominant mutations in SCN5A, which encodes the cardiac sodium channel alpha-subunit (Na_v1.5). To date some SCN5A mutations related with BrS have been identified in voltage sensor of Na_v1.5. Here, we describe a dominant missense mutation (R1629Q) localized in the fourth segment of domain IV region (DIV-S4) in a Chinese Han family. The mutation was identified by direct sequencing of SCN5A from the proband’s DNA. Co-expression of Wild-type (WT) or R1629Q Na_v1.5 channel and hβ1 subunit were achieved in human embryonic kidney cells by transient transfection. Sodium currents were recorded using whole cell patch-clamp protocols. No significant changes between WT and R1629Q currents were observed in current density or steady-state activation. However, hyperpolarized shift of steady–state inactivation curve was identified in cells expressing R1629Q channel (WT: V_1/2 = -81.1 ± 1.3 mV, n = 13; R1629Q: V_1/2 = -101.7 ± 1.2 mV, n = 18). Moreover, R1629Q channel showed enhanced intermediate inactivation and prolonged recovery time from inactivation. In summary, this study reveals that R1629Q mutation causes a distinct loss-of-function of the channel due to alter its electrophysiological characteristics, and facilitates our understanding of biophysical mechanisms of BrS.     

Two Nedd4-binding Motifs Underlie Modulation of Sodium Channel Nav1.6 by p38 MAPK

Sodium channel Na_v1.6 is essential for neuronal excitability in central and peripheral nervous systems. Loss-of-function mutations in Na_v1.6 underlie motor disorders, with homozygous-null mutations causing juvenile lethality. Phosphorylation of Na_v1.6 by the stress-induced p38 MAPK at a Pro-Gly-Ser⁵⁵³-Pro motif in its intracellular loop L1 reduces Na_v1.6 current density in a dorsal root ganglion-derived cell line, without changing its gating properties. Phosphorylated Pro-Gly-Ser⁵⁵³-Pro motif is a putative binding site to Nedd4 ubiquitin ligases, and we hypothesized that Nedd4-like ubiquitin ligases may contribute to channel ubiquitination and internalization. We report here that p38 activation in hippocampal neurons from wild-type mice, but not from Scn8a^medtg mice that lack Na_v1.6, reduces tetrodotoxin-S sodium currents, suggesting isoform-specific modulation of Na_v1.6 by p38 in these neurons. Pharmacological block of endocytosis completely abolishes p38-mediated Na_v1.6 current reduction, supporting our hypothesis that channel internalization underlies current reduction. We also report that the ubiquitin ligase Nedd4-2 interacts with Na_v1.6 via a Pro-Ser-Tyr¹⁹⁴⁵ motif in the C terminus of the channel and reduces Na_v1.6 current density, and we show that this regulation requires both the Pro-Gly-Ser-Pro motif in L1 and the Pro-Ser-Tyr motif in the C terminus. Similarly, both motifs are necessary for p38-mediated reduction of Na_v1.6 current, whereas abrogating binding of the ubiquitin ligase Nedd4-2 to the Pro-Ser-Tyr motif results in stress-mediated increase in Na_v1.6 current density. Thus, phosphorylation of the Pro-Gly-Ser-Pro motif within L1 of Na_v1.6 is necessary for stress-induced current modulation, with positive or negative regulation depending upon the availability of the C-terminal Pro-Ser-Tyr motif to bind Nedd4-2.     

Interactions among DIV voltage-sensor movement,fast inactivation,and resurgent Na current induced by the NaVβ4 open-channel blocking peptide

Resurgent Na current flows as voltage-gated Na channels recover through open states from block by an endogenous open-channel blocking protein, such as the Na_Vβ4 subunit. The open-channel blocker and fast-inactivation gate apparently compete directly, as slowing the onset of fast inactivation increases resurgent currents by favoring binding of the blocker. Here, we tested whether open-channel block is also sensitive to deployment of the DIV voltage sensor, which facilitates fast inactivation. We expressed Na_V1.4 channels in HEK293t cells and assessed block by a free peptide replicating the cytoplasmic tail of Na_Vβ4 (the “β4 peptide”). Macroscopic fast inactivation was disrupted by mutations of DIS6 (L443C/A444W; “CW” channels), which reduce fast-inactivation gate binding, and/or by the site-3 toxin ATX-II, which interferes with DIV movement. In wild-type channels, the β4 peptide competed poorly with fast inactivation, but block was enhanced by ATX. With the CW mutation, large peptide-induced resurgent currents were present even without ATX, consistent with increased open-channel block upon depolarization and slower deactivation after blocker unbinding upon repolarization. The addition of ATX greatly increased transient current amplitudes and further enlarged resurgent currents, suggesting that pore access by the blocker is actually decreased by full deployment of the DIV voltage sensor. ATX accelerated recovery from block at hyperpolarized potentials, however, suggesting that the peptide unbinds more readily when DIV voltage-sensor deployment is disrupted. These results are consistent with two open states in Na channels, dependent on the DIV voltage-sensor position, which differ in affinity for the blocking protein.     

Effect of Alkali Metal Cations on Slow Inactivation of Cardiac Na+ Channels

Human heart Na⁺ channels were expressed transiently in both mammalian cells and Xenopus oocytes, and Na⁺ currents measured using 150 mM intracellular Na⁺. The kinetics of decaying outward Na⁺ current in response to 1-s depolarizations in the F1485Q mutant depends on the predominant cation in the extracellular solution, suggesting an effect on slow inactivation. The decay rate is lower for the alkali metal cations Li⁺, Na⁺, K⁺, Rb⁺, and Cs⁺ than for the organic cations Tris, tetramethylammonium, N-methylglucamine, and choline. In whole cell recordings, raising [Na⁺]_o from 10 to 150 mM increases the rate of recovery from slow inactivation at −140 mV, decreases the rate of slow inactivation at relatively depolarized voltages, and shifts steady-state slow inactivation in a depolarized direction. Single channel recordings of F1485Q show a decrease in the number of blank (i.e., null) records when [Na⁺]_o is increased. Significant clustering of blank records when depolarizing at a frequency of 0.5 Hz suggests that periods of inactivity represent the sojourn of a channel in a slow-inactivated state. Examination of the single channel kinetics at +60 mV during 90-ms depolarizations shows that neither open time, closed time, nor first latency is significantly affected by [Na⁺]_o. However raising [Na⁺]_o decreases the duration of the last closed interval terminated by the end of the depolarization, leading to an increased number of openings at the depolarized voltage. Analysis of single channel data indicates that at a depolarized voltage a single rate constant for entry into a slow-inactivated state is reduced in high [Na⁺]_o, suggesting that the binding of an alkali metal cation, perhaps in the ion-conducting pore, inhibits the closing of the slow inactivation gate.