The Na channel voltage sensor associated with inactivation is localized to the external charged residues of domain IV, S4.

Site-3 toxins have been shown to inhibit a component of gating charge (33% of maximum gating charge, Q(max)) in native cardiac Na channels that has been identified with the open-to-inactivated state kinetic transition. To investigate the role of the three outermost arginine amino acid residues in segment 4 domain IV (R1, R2, R3) in gating charge inhibited by site-3 toxins, we recorded ionic and gating currents from human heart Na channels with mutations of the outermost arginines (R1C, R1Q, R2C, and R3C) expressed in fused, mammalian tsA201 cells. All four mutations had ionic currents that activated over the same voltage range with slope factors of their peak conductance-voltage (G-V) relationships similar to those of wild-type channels, although decay of I(Na) was slowest for R1C and R1Q mutant channels and fastest for R3C mutant channels. After Na channel modification by Ap-A toxin, decays of I(Na) were slowed to similar values for all four channel mutants. Toxin modification produced a graded effect on gating charge (Q) of mutant channels, reducing Q(max) by 12% for the R1C and R1Q mutants, by 22% for the R2C mutant, and by 27% for the R3C mutant, only slightly less than the 31% reduction seen for wild-type currents. Consistent with these findings, the relationship of Q(max) to G(max) was significantly shallower for R1 mutants than for R2C and R3C mutant Na channels. These data suggest that site-3 toxins primarily inhibit gating charge associated with movement of the S4 in domain IV, and that the outermost arginine contributes the largest amount to channel gating, with other arginines contributing less.     

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.     

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.     

Reduced voltage dependence of inactivation in the SCN5A sodium channel mutation delF1617

Deletion of a phenylalanine at position 1617 (delF1617) in the extracellular linker between segments S3 and S4 in domain IV of the human heart Na(+) channel (hH1a) has been tentatively associated with long QT syndrome type 3 (LQT3). In a mammalian cell expression system, we compared whole cell, gating, and single-channel currents of delF1617 with those of wild-type hH1a. The half points of the peak activation-voltage curve for the two channels were similar, as were the deactivation time constants at hyperpolarized test potentials. However, delF1617 demonstrated a significant negative shift of -7 mV in the half point of the voltage-dependent Na(+) channel availability curve compared with wild type. In addition, both the time course of decay of Na(+) current (I(Na)) and two-pulse development of inactivation of delF1617 were faster at negative test potentials, whereas they tended to be slower at positive potentials compared with wild type. Mean channel open times for delF1617 were shorter at potentials <0 mV, whereas they were longer at potentials >0 mV compared with wild type. Using anthopleurin-A, a site-3 toxin that inhibits movement of segment S4 in domain IV (S4-DIV), we found that gating charge contributed by the S4-DIV in delF1617 was reduced 37% compared with wild type. We conclude that deletion of a single amino acid in the S3-S4 linker of domain IV alters the voltage dependence of fast inactivation via a reduction in the gating charge contributed by S4-DIV and can cause either a gain or loss of I(Na), depending on membrane potential.     

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.     

Mechanism of inverted activation of ClC-1 channels caused by a novel myotonia congenita mutation

The voltage-gated chloride channel ClC-1 is the major contributor of membrane conductance in skeletal muscle and has been associated with the inherited muscular disorder myotonia congenita. Here, we report a novel mutation identified in a recessive myotonia congenita family. This mutation, Gly-499 to Arg (G499R) is located in the putative transmembrane domain 10 of the ClC-1 protein. In contrast to normal ClC-1 channels that deactivate upon hyperpolarization, functional expression of G499R ClC-1 yielded a hyperpolarization-activated chloride current when measured in the presence of a high (134 mM) intracellular chloride concentration. Current was abolished when measured with a physiological chloride transmembrane gradient. Electrophysiological analysis of other Gly-499 mutants (G499K, G499Q, and G499E) suggests that the positive charge introduced by the G499R mutation may be responsible for this unique gating behavior. To further explore the function of domain 10, we mutated two charged residues near Gly-499 of ClC-1. Functional analyses of R496Q, R496Q/G499R, R496K, and E500Q mutant channels suggest that the charged residues in domain 10 are important for normal channel function. Study of these mutants may shed further light on the structure and voltage-gating of this channel.     

Neutralization of gating charges in domain II of the sodium channel alpha subunit enhances voltage-sensor trapping by a beta-scorpion toxin

beta-Scorpion toxins shift the voltage dependence of activation of sodium channels to more negative membrane potentials, but only after a strong depolarizing prepulse to fully activate the channels. Their receptor site includes the S3-S4 loop at the extracellular end of the S4 voltage sensor in domain II of the alpha subunit. Here, we probe the role of gating charges in the IIS4 segment in beta-scorpion toxin action by mutagenesis and functional analysis of the resulting mutant sodium channels. Neutralization of the positively charged amino acid residues in the IIS4 segment by mutation to glutamine shifts the voltage dependence of channel activation to more positive membrane potentials and reduces the steepness of voltage-dependent gating, which is consistent with the presumed role of these residues as gating charges. Surprisingly, neutralization of the gating charges at the outer end of the IIS4 segment by the mutations R850Q, R850C, R853Q, and R853C markedly enhances beta-scorpion toxin action, whereas mutations R856Q, K859Q, and K862Q have no effect. In contrast to wild-type, the beta-scorpion toxin Css IV causes a negative shift of the voltage dependence of activation of mutants R853Q and R853C without a depolarizing prepulse at holding potentials from -80 to -140 mV. Reaction of mutant R853C with 2-aminoethyl methanethiosulfonate causes a positive shift of the voltage dependence of activation and restores the requirement for a depolarizing prepulse for Css IV action. Enhancement of sodium channel activation by Css IV causes large tail currents upon repolarization, indicating slowed deactivation of the IIS4 voltage sensor by the bound toxin. Our results are consistent with a voltage-sensor-trapping model in which the beta-scorpion toxin traps the IIS4 voltage sensor in its activated position as it moves outward in response to depolarization and holds it there, slowing its inward movement on deactivation and enhancing subsequent channel activation. Evidently, neutralization of R850 and R853 removes kinetic barriers to binding of the IIS4 segment by Css IV, and thereby enhances toxin-induced channel activation.     

Role of charged residues in the S1-S4 voltage sensor of BK channels

The activation of large conductance Ca(2+)-activated (BK) potassium channels is weakly voltage dependent compared to Shaker and other voltage-gated K(+) (K(V)) channels. Yet BK and K(V) channels share many conserved charged residues in transmembrane segments S1-S4. We mutated these residues individually in mSlo1 BK channels to determine their role in voltage gating, and characterized the voltage dependence of steady-state activation (P(o)) and I(K) kinetics (tau(I(K))) over an extended voltage range in 0-50 microM [Ca(2+)](i). mSlo1 contains several positively charged arginines in S4, but only one (R213) together with residues in S2 (D153, R167) and S3 (D186) are potentially voltage sensing based on the ability of charge-altering mutations to reduce the maximal voltage dependence of P(O). The voltage dependence of P(O) and tau(I(K)) at extreme negative potentials was also reduced, implying that the closed-open conformational change and voltage sensor activation share a common source of gating charge. Although the position of charged residues in the BK and K(V) channel sequence appears conserved, the distribution of voltage-sensing residues is not. Thus the weak voltage dependence of BK channel activation does not merely reflect a lack of charge but likely differences with respect to K(V) channels in the position and movement of charged residues within the electric field. Although mutation of most sites in S1-S4 did not reduce gating charge, they often altered the equilibrium constant for voltage sensor activation. In particular, neutralization of R207 or R210 in S4 stabilizes the activated state by 3-7 kcal mol(-1), indicating a strong contribution of non-voltage-sensing residues to channel function, consistent with their participation in state-dependent salt bridge interactions. Mutations in S4 and S3 (R210E, D186A, and E180A) also unexpectedly weakened the allosteric coupling of voltage sensor activation to channel opening. The implications of our findings for BK channel voltage gating and general mechanisms of voltage sensor activation are discussed.     

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.     

Novel interactions identified between micro -Conotoxin and the Na+ channel domain I P-loop: implications for toxin-pore binding geometry

micro -Conotoxins ( micro -CTX) are peptides that inhibit Na(+) flux by blocking the Na(+) channel pore. Toxin residue arginine 13 is critical for both high affinity binding and for complete block of the single channel current, prompting the simple conventional view that residue 13 (R13) leads toxin docking by entering the channel along the pore axis. To date, the strongest interactions identified are between micro -CTX and domain II (DII) or DIII pore residues of the rat skeletal muscle (Na(v)1.4) Na(+) channels, but little data is available for the role of the DI P-loop in micro -CTX binding due to the lack of critical determinants identified in this domain. Despite being an essential determinant of isoform-specific tetrodotoxin sensitivity, the DI-Y401C variant had little effect on micro -CTX block. Here we report that the charge-changing substitution Y401K dramatically reduced the micro -CTX affinity ( approximately 300-fold). Using mutant cycle analysis, we demonstrate that K401 couples strongly to R13 (DeltaDeltaG > 3.0 kcal/mol) but not R1, K11, or R14 (<1 kcal/mol). Unlike K401, however, a significant coupling was detected between toxin residue 14 and DI-E403K (DeltaDeltaG = 1.4 kcal/mol for the E403K-Q14D pair). This appears to underlie the ability of DI-E403K channels to discriminate between the GIIIA and GIIIB isoforms of micro -CTX (p < 0.05), whereas Y401K, DII-E758Q, and DIII-D1241K do not. We also identify five additional, novel toxin-channel interactions (>0.75 kcal/mol) in DII (E758-K16, D762-R13, D762-K16, E765-R13, E765-K16). Considered together, these new interactions suggest that the R13 side chain and the bulk of the bound toxin micro -CTX molecule may be significantly tilted with respect to pore axis.     

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.     

Effects of the Enantiomers of BayK 8644 on the Charge Movement of L-type Ca Channels in Guinea-pig Ventricular Myocytes

The effects of the agonist enantiomer S(-)Bay K 8644 on gating charge of L-type Ca channels were studied in single ventricular myocytes. From a holding potential (Vh) of -40 mV, saturating (250 nm) S(-)Bay K shifted the half-distribution voltage of the activation charge (Q1) vs. V curve -7.5 +/- 0.8 mV, almost identical to the shift produced in the Ba conductance vs. V curve (-7.7 +/- 2 mV). The maximum Q1 was reduced by 1.7 +/- 0.2 nC/microF, whereas Q2 (charge moved in inactivated channels) was increased in a similar amount (1.4 +/- 0.4 nC/microF). The steady-state availability curves for Q1, Q2, and Ba current showed almost identical negative shifts of -14.8 +/- 1.7 mV, -18.6 +/- 5.8 mV, and -15.2 +/- 2.7 mV, respectively. The effects of the antagonist enantiomer R(+)BayK 8644 were also studied, the Q1 vs. V curve was not significantly shifted, but Q1max (Vh = -40 mV) was reduced and the Q1 availability curve shifted by -24.6 +/- 1.2 mV. We concluded that: a) the left shift in the Q1 vs. V activation curve produced by S(-)BayK is a purely agonistic effect; b) S(-)BayK induced a significantly larger negative shift in the availability curve than in the Q1 vs. V relation, consistent with a direct promotion of inactivation; c) as expected for a more potent antagonist, R(+)Bay K induced a significantly larger negative shift in the availability curve than did S(-)Bay K.     

Clockwise domain arrangement of the sodium channel revealed by (mu)-conotoxin (GIIIA) docking orientation

mu-Conotoxins (mu-CTXs) specifically inhibit Na(+) flux by occluding the pore of voltage-gated Na(+) channels. Although the three-dimensional structures of mu-CTXs are well defined, the molecular configuration of the channel receptor is much less certain; even the fundamental question of whether the four homologous Na(+) channel domains are arranged in a clockwise or counter-clockwise configuration remains unanswered. Residues Asp(762) and Glu(765) from domain II and Asp(1241) from domain III of rat skeletal muscle Na(+) channels are known to be critical for mu-CTX binding. We probed toxin-channel interactions by determining the potency of block of wild-type, D762K, E765K, and D1241C channels by wild-type and point-mutated mu-CTXs (R1A, Q14D, K11A, K16A, and R19A). Individual interaction energies for different toxin-channel pairs were quantified from the half-blocking concentrations using mutant cycle analysis. We find that Asp(762) and Glu(765) interact strongly with Gln(14) and Arg(19) but not Arg(1) and that Asp(1241) is tightly coupled to Lys(16) but not Arg(1) or Lys(11). These newly identified toxin-channel interactions within adjacent domains, interpreted in light of the known asymmetric toxin structure, fix the orientation of the toxin with respect to the channel and reveal that the four internal domains of Na(+) channels are arranged in a clockwise configuration as viewed from the extracellular surface.     

R1 in the Shaker S4 occupies the gating charge transfer center in the resting state

During voltage-dependent activation in Shaker channels, four arginine residues in the S4 segment (R1-R4) cross the transmembrane electric field. It has been proposed that R1-R4 movement is facilitated by a "gating charge transfer center" comprising a phenylalanine (F290) in S2 plus two acidic residues, one each in S2 and S3. According to this proposal, R1 occupies the charge transfer center in the resting state, defined as the conformation in which S4 is maximally retracted toward the cytoplasm. However, other evidence suggests that R1 is located extracellular to the charge transfer center, near I287 in S2, in the resting state. To investigate the resting position of R1, we mutated I287 to histidine (I287H), paired it with histidine mutations of key voltage sensor residues, and determined the effect of extracellular Zn(2+) on channel activity. In I287H+R1H, Zn(2+) generated a slow component of activation with a maximum amplitude (A(slow,max)) of ～56%, indicating that only a fraction of voltage sensors can bind Zn(2+) at a holding potential of -80 mV. A(slow,max) decreased after applying either depolarizing or hyperpolarizing prepulses from -80 mV. The decline of A(slow,max) after negative prepulses indicates that R1 moves inward to abolish ion binding, going beyond the point where reorientation of the I287H and R1H side chains would reestablish a binding site. These data support the proposal that R1 occupies the charge transfer center upon hyperpolarization. Consistent with this, pairing I287H with A359H in the S3-S4 loop generated a Zn(2+)-binding site. At saturating concentrations, A(slow,max) reached 100%, indicating that Zn(2+) traps the I287H+A359H voltage sensor in an absorbing conformation. Transferring I287H+A359H into a mutant background that stabilizes the resting state significantly enhanced Zn(2+) binding at -80 mV. Our results strongly support the conclusion that R1 occupies the gating charge transfer center in the resting conformation.     

Point mutations in alpha-subunit of human cardiac Na+ channels alter Na+ current kinetics

Dietary polyunsaturated fatty acids (PUFAs) prevent ischemia-induced fatal cardiac arrhythmias in animals and probably in humans. This action results from inhibition of ion currents for Na+, Ca2+, and possibly other ions. To extend understanding of this protection we are seeking a possible binding site for the PUFAs on the alpha-subunit of the human cardiac Na+ channel, hH1alpha, transiently expressed in HEK293t cells. Three mutated single amino acid substitutions with lysine were made in the alpha-subunit at Domain 4-Segment 6 (D4-S6) for F1760, Y1767 and at D1-S6 for N406. These are in the putative sites of binding of local anesthetics and batrachotoxin, respectively. The mutants F1760K, Y1767K, and N406K, separately and to different extents, affected the current density, the steady-state inactivation potential, accelerated inactivation, delayed recovery from inactivation, and affected voltage-dependent block, but did not affect activation of the hH1alpha. It is essential to learn that single point mutations in D1-S6 and D4-S6 alone significantly modify the kinetics of human cardiac hH1alpha Na+ currents. The effects of PUFAs on these mutant channels will be the subject of subsequent reports.     

Charge conversion enables quantification of the proximity between a normally-neutral mu-conotoxin (GIIIA) site and the Na+ channel pore

mu-Conotoxin (mu-CTX) inhibits Na+ flux by obstructing the Na+ channel pore. Previous studies of mu-CTX have focused only on charged toxin residues, ignoring the neutral sites. Here we investigated the proximity between the C-terminal neutral alanine (A22) of mu-CTX and the Na+ channel pore by replacing it with the negatively charged glutamate. The analog A22E and wild-type (WT) mu-CTX exhibited identical nuclear magnetic resonance spectra except at the site of replacement, verifying that they have identical backbone structures. A22E significantly reduced mu-CTX affinity for WT mu1 Na+ channels (90-fold), as if the inserted glutamate repels the anionic pore receptor. We then looked for the interacting partner(s) of residue 22 by determining the potency of block of Y401K, Y401A, E758Q, D762K, D762A, E765K, E765A and D1241K channels by WT mu-CTX and A22E, followed by mutant cycle analysis to assess their individual couplings. Our results show that A22E interacts strongly with E765K from domain II (DII) (deltadeltaG=2.2 +/- 0.1 vs. <1 kcal/mol for others). We conclude that mu-CTX residue 22 closely associates with the DII pore in the toxin-bound channel complex. The approach taken may be further exploited to study the proximity of other neutral toxin residues with the Na+ channel pore.     

Two basic residues of the h-VPAC1 receptor second transmembrane helix are essential for ligand binding and signal transduction

We mutated the vasoactive intestinal peptide (VIP) Asp(3) residue and two VPAC(1) receptor second transmembrane helix basic residues (Arg(188) and Lys(195)). VIP had a lower affinity for R188Q, R188L, K195Q, and K195I VPAC(1) receptors than for VPAC(1) receptors. [Asn(3)] VIP and [Gln(3)] VIP had lower affinities than VIP for VPAC(1) receptors but higher affinities for the mutant receptors; the two basic amino acids facilitated the introduction of the negatively charged aspartate inside the transmembrane domain. The resulting interaction was necessary for receptor activation. 1/[Asn(3)] VIP and [Gln(3)] VIP were partial agonists at VPAC(1) receptors; 2/VIP did not fully activate the K195Q, K195I, R188Q, and R188L VPAC(1) receptors; a VIP analogue ([Arg(16)] VIP) was more efficient than VIP at the four mutated receptors; and [Asn(3)] VIP and [Gln(3)] VIP were more efficient than VIP at the R188Q and R188L VPAC(1) receptors; 3/the [Asp(3)] negative charge did not contribute to the recognition of the VIP(1) antagonist, [AcHis(1),D-Phe(2),Lys(15),Arg(16),Leu(27)] VIP ()/growth hormone releasing factor (8-27). This is the first demonstration that, to activate the VPAC(1) receptor, the Asp(3) side chain of VIP must penetrate within the transmembrane domain, in close proximity to two highly conserved basic amino acids from transmembrane 2.     

Differential phospholipid binding by site 3 and site 4 toxins. Implications for structural variability between voltage-sensitive sodium channel domains

It has been shown recently that polypeptide toxins that modulate the gating properties of voltage-sensitive cation channels are able to bind to phospholipid membranes, leading to the suggestion that these toxins are able to access a channel-binding site that remains membrane-restricted (Lee, S.-Y., and MacKinnon, R. (2004) Nature 430, 232-235). We therefore examined the ability of anthopleurin B (ApB), a sea anemone toxin that selectively modifies inactivation kinetics of Na(V)1.x channels, and ProTx-II, a spider toxin that modifies activation kinetics of the same channels, to bind to liposomes. Whereas ProTx-II can be quantitatively depleted from solution upon incubation with phosphatidylcholine/phosphatidylserine liposomes, ApB displays no discernible phospholipid binding activity. We therefore examined the activities of structurally unrelated site 3 and site 4 toxins derived from Leiurus and Centruroides venoms, respectively, in the same assay. Like ApB, the site 3 toxin LqqV shows no lipid binding activity, whereas the site 4 toxin Centruroides toxin II, like ProTx-II, is completely bound. We conclude that toxins that modify inactivation kinetics via binding to Na(V)1.x site 3 lack the ability to bind phospholipids, whereas site 4 toxins, which modify activation, have this activity. This inherent difference suggests that the conformation of domain II more closely resembles that of the K(V)AP channel than does the conformation of domain IV.     

A Na+ channel mutation linked to hypokalemic periodic paralysis exposes a proton-selective gating pore

The heritable muscle disorder hypokalemic periodic paralysis (HypoPP) is characterized by attacks of flaccid weakness, brought on by sustained sarcolemmal depolarization. HypoPP is genetically linked to missense mutations at charged residues in the S4 voltage-sensing segments of either CaV1.1 (the skeletal muscle L-type Ca(2+) channel) or NaV1.4 (the skeletal muscle voltage-gated Na(+) channel). Although these mutations alter the gating of both channels, these functional defects have proven insufficient to explain the sarcolemmal depolarization in affected muscle. Recent insight into the topology of the S4 voltage-sensing domain has aroused interest in an alternative pathomechanism, wherein HypoPP mutations might generate an aberrant ionic leak conductance by unblocking the putative aqueous crevice ("gating-pore") in which the S4 segment resides. We tested the rat isoform of NaV1.4 harboring the HypoPP mutation R663H (human R669H ortholog) at the outermost arginine of S4 in domain II for a gating-pore conductance. We found that the mutation R663H permits transmembrane permeation of protons, but not larger cations, similar to the conductance displayed by histidine substitution at Shaker K(+) channel S4 sites. These results are consistent with the notion that the outermost charged residue in the DIIS4 segment is simultaneously accessible to the cytoplasmic and extracellular spaces when the voltage sensor is positioned inwardly. The predicted magnitude of this proton leak in mature skeletal muscle is small relative to the resting K(+) and Cl(-) conductances, and is thus not likely to fully account for the aberrant sarcolemmal depolarization underlying the paralytic attacks. Rather, it is possible that a sustained proton leak may contribute to instability of V(REST) indirectly, for instance, by interfering with intracellular pH homeostasis.     

Non-Native R1 Substitution in the S4 Domain Uniquely Alters Kv4.3 Channel Gating

The S4 transmembrane domain in Shaker (Kv1) voltage-sensitive potassium channels has four basic residues (R1–R4) that are responsible for carrying the majority of gating charge. In Kv4 channels, however, R1 is replaced by a neutral valine at position 287. Among other differences, Kv4 channels display prominent closed state inactivation, a mechanism which is minimal in Shaker. To determine if the absence of R1 is responsible for important variation in gating characteristics between the two channel types, we introduced the V287R mutant into Kv4.3 and analyzed its effects on several voltage sensitive gating transitions. We found that the mutant increased the voltage sensitivity of steady-state activation and altered the kinetics of activation and deactivation processes. Although the kinetics of macroscopic inactivation were minimally affected, the characteristics of closed-state inactivation and recovery from open and closed inactivated states were significantly altered. The absence of R1 can only partially account for differences in the effective voltage sensitivity of gating between Shaker and Kv4.3. These results suggest that the S4 domain serves an important functional role in Kv4 channel activation and deactivation processes, and also those of closed-state inactivation and recovery.