Molecular action of lidocaine on the voltage sensors of sodium channels

Block of sodium ionic current by lidocaine is associated with alteration of the gating charge-voltage (Q-V) relationship characterized by a 38% reduction in maximal gating charge (Q(max)) and by the appearance of additional gating charge at negative test potentials. We investigated the molecular basis of the lidocaine-induced reduction in cardiac Na channel-gating charge by sequentially neutralizing basic residues in each of the voltage sensors (S4 segments) in the four domains of the human heart Na channel (hH1a). By determining the relative reduction in the Q(max) of each mutant channel modified by lidocaine we identified those S4 segments that contributed to a reduction in gating charge. No interaction of lidocaine was found with the voltage sensors in domains I or II. The largest inhibition of charge movement was found for the S4 of domain III consistent with lidocaine completely inhibiting its movement. Protection experiments with intracellular MTSET (a charged sulfhydryl reagent) in a Na channel with the fourth outermost arginine in the S4 of domain III mutated to a cysteine demonstrated that lidocaine stabilized the S4 in domain III in a depolarized configuration. Lidocaine also partially inhibited movement of the S4 in domain IV, but lidocaine's most dramatic effect was to alter the voltage-dependent charge movement of the S4 in domain IV such that it accounted for the appearance of additional gating charge at potentials near -100 mV. These findings suggest that lidocaine's actions on Na channel gating charge result from allosteric coupling of the binding site(s) of lidocaine to the voltage sensors formed by the S4 segments in domains III and IV.     

Tracking voltage-dependent conformational changes in skeletal muscle sodium channel during activation

The primary voltage sensor of the sodium channel is comprised of four positively charged S4 segments that mainly differ in the number of charged residues and are expected to contribute differentially to the gating process. To understand their kinetic and steady-state behavior, the fluorescence signals from the sites proximal to each of the four S4 segments of a rat skeletal muscle sodium channel were monitored simultaneously with either gating or ionic currents. At least one of the kinetic components of fluorescence from every S4 segment correlates with movement of gating charge. The fast kinetic component of fluorescence from sites S216C (S4 domain I), S660C (S4 domain II), and L1115C (S4 domain III) is comparable to the fast component of gating currents. In contrast, the fast component of fluorescence from the site S1436C (S4 domain IV) correlates with the slow component of gating. In all the cases, the slow component of fluorescence does not have any apparent correlation with charge movement. The fluorescence signals from sites reflecting the movement of S4s in the first three domains initiate simultaneously, whereas the fluorescence signals from the site S1436C exhibit a lag phase. These results suggest that the voltage-dependent movement of S4 domain IV is a later step in the activation sequence. Analysis of equilibrium and kinetic properties of fluorescence over activation voltage range indicate that S4 domain III is likely to move at most hyperpolarized potentials, whereas the S4s in domain I and domain II move at more depolarized potentials. The kinetics of fluorescence changes from sites near S4-DIV are slower than the activation time constants, suggesting that the voltage-dependent movement of S4-DIV may not be a prerequisite for channel opening. These experiments allow us to map structural features onto the kinetic landscape of a sodium channel during activation.     

The role of the putative inactivation lid in sodium channel gating current immobilization

We investigated the contribution of the putative inactivation lid in voltage-gated sodium channels to gating charge immobilization (i.e., the slow return of gating charge during repolarization) by studying a lid-modified mutant of the human heart sodium channel (hH1a) that had the phenylalanine at position 1485 in the isoleucine, phenylalanine, and methionine (IFM) region of the domain III-IV linker mutated to a cysteine (ICM-hH1a). Residual fast inactivation of ICM-hH1a in fused tsA201 cells was abolished by intracellular perfusion with 2.5 mM 2-(trimethylammonium)ethyl methanethiosulfonate (MTSET). The time constants of gating current relaxations in response to step depolarizations and gating charge-voltage relationships were not different between wild-type hH1a and ICM-hH1a(MTSET). The time constant of the development of charge immobilization assayed at -180 mV after depolarization to 0 mV was similar to the time constant of inactivation of I(Na) at 0 mV for hH1a. By 44 ms, 53% of the gating charge during repolarization returned slowly; i.e., became immobilized. In ICM-hH1a(MTSET), immobilization occurred with a similar time course, although only 31% of gating charge upon repolarization (OFF charge) immobilized. After modification of hH1a and ICM-hH1a(MTSET) with Anthopleurin-A toxin, a site-3 peptide toxin that inhibits movement of the domain IV-S4, charge immobilization did not occur for conditioning durations up to 44 ms. OFF charge for both hH1a and ICM-hH1a(MTSET) modified with Anthopleurin-A toxin were similar in time course and in magnitude to the fast component of OFF charge in ICM-hH1a(MTSET) in control. We conclude that movement of domain IV-S4 is the rate-limiting step during repolarization, and it contributes to charge immobilization regardless of whether the inactivation lid is bound. Taken together with previous reports, these data also suggest that S4 in domain III contributes to charge immobilization only after binding of the inactivation lid.     

Alpha-scorpion toxin impairs a conformational change that leads to fast inactivation of muscle sodium channels

Alpha-scorpion toxins bind in a voltage-dependent way to site 3 of the sodium channels, which is partially formed by the loop connecting S3 and S4 segments of domain IV, slowing down fast inactivation. We have used Ts3, an alpha-scorpion toxin from the Brazilian scorpion Tityus serrulatus, to analyze the effects of this family of toxins on the muscle sodium channels expressed in Xenopus oocytes. In the presence of Ts3 the total gating charge was reduced by 30% compared with control conditions. Ts3 accelerated the gating current kinetics, decreasing the contribution of the slow component to the ON gating current decay, indicating that S4-DIV was specifically inhibited by the toxin. In addition, Ts3 accelerated and decreased the fraction of charge in the slow component of the OFF gating current decay, which reflects an acceleration in the recovery from the fast inactivation. Site-specific fluorescence measurements indicate that Ts3 binding to the voltage-gated sodium channel eliminates one of the components of the fluorescent signal from S4-DIV. We also measured the fluorescent signals produced by the movement of the first three voltage sensors to test whether the bound Ts3 affects the movement of the other voltage sensors. While the fluorescence-voltage (F-V) relationship of domain II was only slightly affected and the F-V of domain III remained unaffected in the presence of Ts3, the toxin significantly shifted the F-V of domain I to more positive potentials, which agrees with previous studies showing a strong coupling between domains I and IV. These results are consistent with the proposed model, in which Ts3 specifically impairs the fraction of the movement of the S4-DIV that allows fast inactivation to occur at normal rates.     

The outermost lysine in the S4 of domain III contributes little to the gating charge in sodium channels

We investigated the contribution the four outermost basic residues (K1, R2, R3, R4) in segment 4 of domain III in the human cardiac Na channel (hH1a, Na(V)1.5) to the total gating charge (Q(max)). Each of the four basic residues were mutated individually to a cysteine. In addition, R2 was also mutated to a glutamate. All mutant channels were transiently expressed with the alpha1 subunit in fused tsA201 cells. We used the relative reduction in Q(max) caused by anthopleurin-A (ApA) toxin, a site-3 toxin known to inhibit the movement of gating charge associated with domain IV, to estimate the size of the contribution from each basic residue. Studies of the toxin's ability to inhibit gating charge in mutant channels showed that R2 contributed 19-20% to the Q(max), R3 contributed 10%, and K1 and R4 made almost no contribution. In contrast to the outermost basic residue in the S4 of Shaker K channels and in the S4 of domain IV in hH1a, the outermost charge (K1) in domain III of Na channels is outside the voltage field.     

Isoform-specific lidocaine block of sodium channels explained by differences in gating

When depolarized from typical resting membrane potentials (V(rest) approximately -90 mV), cardiac sodium (Na) currents are more sensitive to local anesthetics than brain or skeletal muscle Na currents. When expressed in Xenopus oocytes, lidocaine block of hH1 (human cardiac) Na current greatly exceeded that of mu1 (rat skeletal muscle) at membrane potentials near V(rest), whereas hyperpolarization to -140 mV equalized block of the two isoforms. Because the isoform-specific tonic block roughly parallels the drug-free voltage dependence of channel availability, isoform differences in the voltage dependence of fast inactivation could underlie the differences in block. However, after a brief (50 ms) depolarizing pulse, recovery from lidocaine block is similar for the two isoforms despite marked kinetic differences in drug-free recovery, suggesting that differences in fast inactivation cannot entirely explain the isoform difference in lidocaine action. Given the strong coupling between fast inactivation and other gating processes linked to depolarization (activation, slow inactivation), we considered the possibility that isoform differences in lidocaine block are explained by differences in these other gating processes. In whole-cell recordings from HEK-293 cells, the voltage dependence of hH1 current activation was approximately 20 mV more negative than that of mu1. Because activation and closed-state inactivation are positively coupled, these differences in activation were sufficient to shift hH1 availability to more negative membrane potentials. A mutant channel with enhanced closed-state inactivation gating (mu1-R1441C) exhibited increased lidocaine sensitivity, emphasizing the importance of closed-state inactivation in lidocaine action. Moreover, when the depolarization was prolonged to 1 s, recovery from a "slow" inactivated state with intermediate kinetics (I(M)) was fourfold longer in hH1 than in mu1, and recovery from lidocaine block in hH1 was similarly delayed relative to mu1. We propose that gating processes coupled to fast inactivation (activation and slow inactivation) are the key determinants of isoform-specific local anesthetic action.     

An EF-hand in the sodium channel couples intracellular calcium to cardiac excitability

Sodium channels initiate the electrical cascade responsible for cardiac rhythm, and certain life-threatening arrhythmias arise from Na(+) channel dysfunction. We propose a novel mechanism for modulation of Na(+) channel function whereby calcium ions bind directly to the human cardiac Na(+) channel (hH1) via an EF-hand motif in the C-terminal domain. A functional role for Ca(2+) binding was identified electrophysiologically, by measuring Ca(2+)-induced modulation of hH1. A small hH1 fragment containing the EF-hand motif was shown to form a structured domain and to bind Ca(2+) with affinity characteristic of calcium sensor proteins. Mutations in this domain reduce Ca(2+) affinity in vitro and the inactivation gating effects of Ca(2+) in electrophysiology experiments. These studies reveal the molecular basis for certain forms of long QT syndrome and other arrhythmia-producing syndromes, and suggest a potential pharmacological target for antiarrhythmic drug design.     

Characterization of the isoform-specific differences in the gating of neuronal and muscle sodium channels

Human heart (hH1), human skeletal muscle (hSkM1), and rat brain (rIIA) Na channels were expressed in cultured cells and the activation and inactivation of the whole-cell Na currents measured using the patch clamp technique. hH1 Na channels were found to activate and inactivate at more hyperpolarized voltages than hSkM1 and rIIA. The conductance versus voltage and steady state inactivation relationships have midpoints of -48 and -92 mV (hH1), -28 and -72 mV (hSkM1), and -22 and -61 mV (rIIA). At depolarized voltages, where Na channels predominately inactivate from the open state, the inactivation of hH1 is 2-fold slower than that of hSkM1 and rIIA. The recovery from fast inactivation of all three isoforms is well described by a single rapid component with time constants at -100 mV of 44 ms (hH1), 4.7 ms (hSkM1), and 7.6 ms (rIIA). After accounting for differences in voltage dependence, the kinetics of activation, inactivation, and recovery of hH1 were found to be generally slower than those of hSkM1 and rIIA. Modeling of Na channel gating at hyperpolarized voltages where the channel does not open suggests that the slow rate of recovery from inactivation of hH1 accounts for most of the differences in the steady-state inactivation of these Na channels.     

Structural insight into the transmembrane segments 3 and 4 of the hERG potassium channel

The hERG (human ether‐a‐go‐go related gene) potassium channel is a voltage‐gated potassium channel containing an N‐terminal domain, a voltage‐sensor domain, a pore domain and a C‐terminal domain. The transmembrane segment 4 (S4) is important for sensing changes of membrane potentials through positively charge residues. A construct containing partial S2–S3 linker, S3, S4 and the S4–S5 linker of the hERG channel was purified into detergent micelles. This construct exhibits good quality NMR spectrum when it was purified in lyso‐myristoyl phosphatidylglycerol (LMPG) micelles. Structural study showed that S3 contains two short helices with a negatively charged surface. The S4 and S4–S5 linker adopt helical structures. The six positively charged residues in S4 localize at different sides, suggesting that they may have different functions in channel gating. Relaxation studies indicated that S3 is more flexible than S4. The boundaries of S3–S4 and S4–S4–S5 linker were identified. Our results provided structural information of the S3 and S4, which will be helpful to understand their roles in channel gating. Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd.     

Modulation of the Shaker K(+) channel gating kinetics by the S3-S4 linker

In Shaker K(+) channels depolarization displaces outwardly the positively charged residues of the S4 segment. The amount of this displacement is unknown, but large movements of the S4 segment should be constrained by the length and flexibility of the S3-S4 linker. To investigate the role of the S3-S4 linker in the ShakerH4Delta(6-46) (ShakerDelta) K(+) channel activation, we constructed S3-S4 linker deletion mutants. Using macropatches of Xenopus oocytes, we tested three constructs: a deletion mutant with no linker (0 aa linker), a mutant containing a linker 5 amino acids in length, and a 10 amino acid linker mutant. Each of the three mutants tested yielded robust K(+) currents. The half-activation voltage was shifted to the right along the voltage axis, and the shift was +45 mV in the case of the 0 aa linker channel. In the 0 aa linker, mutant deactivation kinetics were sixfold slower than in ShakerDelta. The apparent number of gating charges was 12.6+/-0.6 e(o) in ShakerDelta, 12.7+/-0.5 in 10 aa linker, and 12.3+/-0.9 in 5 aa linker channels, but it was only 5.6+/-0.3 e(o) in the 0 aa linker mutant channel. The maximum probability of opening (P(o)(max)) as measured using noise analysis was not altered by the linker deletions. Activation kinetics were most affected by linker deletions; at 0 mV, the 5 and 0 aa linker channels' activation time constants were 89x and 45x slower than that of the ShakerDelta K(+) channel, respectively. The initial lag of ionic currents when the prepulse was varied from -130 to -60 mV was 0.5, 14, and 2 ms for the 10, 5, and 0 aa linker mutant channels, respectively. These results suggest that: (a) the S4 segment moves only a short distance during activation since an S3-S4 linker consisting of only 5 amino acid residues allows for the total charge displacement to occur, and (b) the length of the S3-S4 linker plays an important role in setting ShakerDelta channel activation and deactivation kinetics.     

Modulation of Nav1.5 channel function by an alternatively spliced sequence in the DII/DIII linker region

In the present study, we identified a novel splice variant of the human cardiac Na(+) channel Na(v)1.5 (Na(v)1.5d), in which a 40-amino acid sequence of the DII/DIII intracellular linker is missing due to a partial deletion of exon 17. Expression of Na(v)1.5d occurred in embryonic and adult hearts of either sex, indicating that the respective alternative splicing is neither age-dependent nor gender-specific. In contrast, Na(v)1.5d was not detected in the mouse heart, indicating that alternative splicing of Na(v)1.5 is species-dependent. In HEK293 cells, splice variant Na(v)1.5d generated voltage-dependent Na(+) currents that were markedly reduced compared with wild-type Na(v)1.5. Experiments with mexiletine and 8-bromo-cyclic AMP suggested that the trafficking of Na(v)1.5d channels was not impaired. However, single-channel recordings showed that the whole-cell current reduction was largely due to a significantly reduced open probability. Additionally, steady-state activation and inactivation were shifted to depolarized potentials by 15.9 and 5.1 mV, respectively. Systematic mutagenesis analysis of the spliced region provided evidence that a short amphiphilic region in the DII/DIII linker resembling an S4 voltage sensor of voltage-gated ion channels is an important determinant of Na(v)1.5 channel gating. Moreover, the present study identified novel short sequence motifs within this amphiphilic region that specifically affect the voltage dependence of steady-state activation and inactivation and current amplitude of human Na(v)1.5.     

Helical secondary structure of the external S3-S4 linker of pacemaker (HCN) channels revealed by site-dependent perturbations of activation phenotype

If, encoded by the hyperpolarization-activated cyclic nucleotide-modulated channel family (HCN1-4), contributes significantly to neuronal and cardiac pacing. Recently, we reported that the S3-S4 residue Glu-235 of HCN1 influences activation by acting as a surface charge. However, it is uncertain whether other residues of the external S3-S4 linker are also involved in gating. Furthermore, the secondary conformation of the linker is not known. Here we probed the structural and functional role of the HCN1 S3-S4 linker by introducing systematic mutations into the entire linker (defined as 229-237) and studying their effects. We found that the mutations K230A (-62.2 +/- 3.4 mV versus -72.2 +/- 1.7 mV of wild type (WT)), G231A (-64.4 +/- 1.3 mV), M232A (V(1/2) = -63.1 +/- 1.1 mV), and E235G (-65.4 +/- 1.5 mV) produced depolarizing activation shifts. Although E229A and M232A decelerated gating kinetics (<13- and 3-fold, respectively), K230A and G231A accelerated both activation and deactivation (< approximately 2-3-fold). D233A, S234A, V236A, and Y237A channels exhibited WT properties (p > 0.05). Shortening the linker (EVY235-237deltadeltadelta) caused depolarizing activation shift and slowed kinetics that could not be explained by removing the charge at position 235 alone. Secondary structural predictions by the modeling algorithms SSpro2 and PROF, along with refinements by our experimental data, suggest that part of the S3-S4 linker conforms a helical structure with the functionally important residues Met-232, Glu-235, and Gly-231 (|deltadeltaG|>1 kcal/mol) clustered on one side.     

Deletion of the S3-S4 linker in the Shaker potassium channel reveals two quenching groups near the outside of S4

When attached outside the voltage-sensing S4 segment of the Shaker potassium channel, the fluorescent probe tetramethylrhodamine (TMRM) undergoes voltage-dependent fluorescence changes (DeltaF) due to differential interaction with a pH-titratable external protein-lined vestibule (Cha, A., and F. Bezanilla. 1998. J. Gen. Physiol. 112:391-408.). We attached TMRM at the same sites [corresponding to M356C and A359C in the wild-type (wt) channel] in a deletion mutant of Shaker where all but the five amino acids closest to S4 had been removed from the S3-S4 linker. In the deletion mutant, the maximal DeltaF/F seen was diminished 10-fold, and the DeltaF at M356C became pH independent, suggesting that the protein-lined vestibule is made up in large part by the S3-S4 linker. The residual DeltaF showed that the probe still interacted with two putative quenching groups near the S4 segment. One group was detected by M356C-TMRM (located outside of S3 in the deletion mutant) and reported on deactivation gating charge movement when applying hyperpolarizing voltage steps from a holding potential of 0 mV. During activating voltage steps from a holding potential of -90 mV, the fluorescence lagged considerably behind the movement of gating charge over a range of potentials. Another putative quenching group was seen by probes attached closer to the S4 and caused a DeltaF at extreme hyperpolarizations (more negative than -90 mV) only. A signal from the interaction with this group in the wt S3-S4 linker channel (at L361C) correlated with gating charge moving in the hyperpolarized part of the Q-V curve. Probe attached at A359C in the deletion mutant and at L361C in wt channel showed a biphasic DeltaF as the probe oscillated between the two groups, revealing that there is a transient state of the voltage sensor in between, where the probe has maximal fluorescence. We conclude that the voltage sensor undergoes two distinct conformational changes as seen from probes attached outside the S4 segment.     

Size matters: Erythromelalgia mutation S241T in Nav1.7 alters channel gating

The Nav1.7 sodium channel is preferentially expressed in most nociceptive dorsal root ganglion neurons and in sympathetic neurons. Inherited erythromelalgia (IEM, also known as erythermalgia), an autosomal dominant neuropathy characterized by burning pain in the extremities in response to mild warmth, has been linked to mutations in Nav1.7. Recently, a substitution of Ser-241 by threonine (S241T) in the domain I S4-S5 linker of Nav1.7 was identified in a family with IEM. To investigate the possible causative role of this mutation in the pathophysiology of IEM, we used whole-cell voltage-clamp analysis to study the effects of S241T on Nav1.7 gating in HEK293 cells. We found a hyperpolarizing shift of activation midpoint by 8.4 mV, an accelerated time to peak, slowing of deactivation, and an increase in the current in response to small, slow depolarizations. Additionally, S241T produced an enhancement of slow inactivation, shifting the midpoint by -12.3 mV. Because serine and threonine have similar biochemical properties, the S241T substitution suggested that the size of the side chain at this position affected channel gating. To test this hypothesis, we investigated the effect of S241A and S241L substitutions on the gating properties of Nav1.7. Although S241A did not alter the properties of the channel, S241L mimicked the effects of S241T. We conclude that the linker between S4 and S5 in domain I of Nav1.7 modulates gating of this channel, and that a larger side chain at position 241 interferes with its gating mechanisms.     

A hot spot for the interaction of gating modifier toxins with voltage-dependent ion channels

The gating modifier toxins are a large family of protein toxins that modify either activation or inactivation of voltage-gated ion channels. omega-Aga-IVA is a gating modifier toxin from spider venom that inhibits voltage-gated Ca(2+) channels by shifting activation to more depolarized voltages. We identified two Glu residues near the COOH-terminal edge of S3 in the alpha(1A) Ca(2+) channel (one in repeat I and the other in repeat IV) that align with Glu residues previously implicated in forming the binding sites for gating modifier toxins on K(+) and Na(+) channels. We found that mutation of the Glu residue in repeat I of the Ca(2+) channel had no significant effect on inhibition by omega-Aga-IVA, whereas the equivalent mutation of the Glu in repeat IV disrupted inhibition by the toxin. These results suggest that the COOH-terminal end of S3 within repeat IV contributes to forming a receptor for omega-Aga-IVA. The strong predictive value of previous mapping studies for K(+) and Na(+) channel toxins argues for a conserved binding motif for gating modifier toxins within the voltage-sensing domains of voltage-gated ion channels.     

Uncoupling of gating charge movement and closure of the ion pore during recovery from inactivation in the Kv1.5 channel

Both wild-type (WT) and nonconducting W472F mutant (NCM) Kv1.5 channels are able to conduct Na(+) in their inactivated states when K(+) is absent. Replacement of K(+) with Na(+) or NMG(+) allows rapid and complete inactivation in both WT and W472F mutant channels upon depolarization, and on return to negative potentials, transition of inactivated channels to closed-inactivated states is the first step in the recovery of the channels from inactivation. The time constant for immobilized gating charge recovery at -100 mV was 11.1 +/- 0.4 ms (n = 10) and increased to 19.0 +/- 1.6 ms (n = 3) when NMG(+)(o) was replaced by Na(+)(o). However, the decay of the Na(+) tail currents through inactivated channels at -100 mV had a time constant of 129 +/- 26 ms (n = 18), much slower than the time required for gating charge recovery. Further experiments revealed that the voltage-dependence of gating charge recovery and of the decay of Na(+) tail currents did not match over a 60 mV range of repolarization potentials. A faster recovery of gating charge than pore closure was also observed in WT Kv1.5 channels. These results provide evidence that the recovery of the gating elements is uncoupled from that of the pore in Na(+)-conducting inactivated channels. The dissociation of the gating charge movements and the pore closure could also be observed in the presence of symmetrical Na(+) but not symmetrical Cs(+). This difference probably stems from the difference in the respective abilities of the two ions to limit inactivation to the P-type state or prevent it altogether.     

Enhancement of closed-state inactivation in long QT syndrome sodium channel mutation DeltaKPQ

DeltaKPQ, a three amino acid [lysine (K), proline (P), glutamine (Q)] deletion mutation of the human cardiac Na channel (hH1), which is one cause of long QT syndrome (LQT3), has impaired inactivation resulting in a late sodium current. To better understand inactivation in DeltaKPQ, we applied a site-3 toxin anthopleurin A, which has been shown to inhibit inactivation from the open state with little or no effect on inactivation from the closed state(s) in wild-type hH1. In contrast to the effect of site-3 toxins on wild-type hH1, inactivation from closed state(s) in toxin-modified DeltaKPQ demonstrated a large negative shift in the Na channel availability curve of nearly -14 mV. Recovery from inactivation showed that toxin-modified DeltaKPQ channels recovered slightly faster than those in control, whereas development of inactivation at potentials negative to -80 mV showed that inactivation developed much more rapidly in toxin-modified DeltaKPQ channels compared with control. An explanation for our results is that closed-state inactivation in toxin-modified DeltaKPQ is enhanced by the mutated inactivation lid being positioned "closer" to its receptor resulting in an increased rate of association between the inactivation lid and its receptor.     

Channel Activation Voltage Alone Is Directly Altered in an Isoform-specific Manner by Na<Subscript>v1.4</Subscript> and Na<Subscript>v1.5</Subscript> Cytoplasmic Linkers

The isoform-specific direct role of cytoplasmic loops in the gating of two voltage-gated sodium channel isoforms, the human cardiac channel (Na_v1.5; hH1) and the human adult skeletal muscle channel (Na_v1.4; hSkM1), was investigated. Comparison of biophysical characteristics was made among hSkM1, hH1, and several hSkM1/hH1 chimeras in which the putative cytoplasmic loops that join domain I to II (loop A) and domain II to III (loop B) from one isoform replaced one or both of the analogous loops from the other isoform. For all parameters measured, hSkM1 and hH1 behavior were significantly different. Comparison of hSkM1 and hH1 biophysical characteristics with the function of their respective chimeras indicate that only the half-activation voltage (V_a) is directly and differently altered by the species of cytoplasmic loop such that a channel consisting of one or both hSkM1 loops activates at smaller depolarizations, while a larger depolarization is required for activation of a channel containing one or both of the analogous hH1 loops. When either cardiac channel loop A or B is attached to hSkM1, a 6–7 mV depolarizing shift in V_a is measured, increasing to a nearly 20 mV depolarization when both cardiac-channel loops are attached. The addition of either skeletal muscle-channel loop to hH1 causes a 7 mV hyperpolarization in V_a, which increases to about 10 mV for the double loop chimera. There is no significant difference in either steady-state inactivation or in the recovery from inactivation data between hSkM1 and its chimeras and between hH1 and its chimeras. Data indicate that the cytoplasmic loops contribute directly to the magnitude of the window current, suggesting that channels containing skeletal muscle loops have three times the peak persistent channel activity compared to channels containing the cardiac loops. An electrostatic mechanism, in which surface charge differences among these loops might alter differently the voltage sensed by the gating mechanism of the channel, can not account for the observed isoform-specific effects of these loops only on channel activation voltage. In summary, although the DI-DII and DII-DIII loop structures among isoforms are not well conserved, these data indicate that only one gating parameter, V_a is affected directly and in an isoform-specific manner by these divergent loop structures, creating loop-specific window currents and percentages of persistently active channels at physiological voltages that will likely impact the excitability of the cell.     

Kv Channel S1-S2 Linker Working as a Binding Site of Human β-Defensin 2 for Channel Activation Modulation

Among the three extracellular domains of the tetrameric voltage-gated K⁺ (Kv) channels consisting of six membrane-spanning helical segments named S1–S6, the functional role of the S1-S2 linker still remains unclear because of the lack of a peptide ligand. In this study, the Kv1.3 channel S1-S2 linker was reported as a novel receptor site for human β-defensin 2 (hBD2). hBD2 shifts the conductance-voltage relationship curve of the human Kv1.3 channel in a positive direction by nearly 10.5 mV and increases the activation time constant for the channel. Unlike classical gating modifiers of toxin peptides from animal venoms, which generally bind to the Kv channel S3-S4 linker, hBD2 only targets residues in both the N and C termini of the S1-S2 linker to influence channel gating and inhibit channel currents. The increment and decrement of the basic residue number in a positively charged S4 sensor of Kv1.3 channel yields conductance-voltage relationship curves in the positive direction by ∼31.2 mV and 2–4 mV, which suggests that positively charged hBD2 is anchored in the channel S1-S2 linker and is modulating channel activation through electrostatic repulsion with an adjacent S4 helix. Together, these findings reveal a novel peptide ligand that binds with the Kv channel S1-S2 linker to modulate channel activation. These findings also highlight the functional importance of the Kv channel S1-S2 linker in ligand recognition and modification of channel activation.