Outward stabilization of the voltage sensor in domain II but not domain I speeds inactivation of voltage-gated sodium channels.

To determine the roles of the individual S4 segments in domains I and II to activation and inactivation kinetics of sodium current (INa) in NaV1.5, we used a tethered biotin and avidin approach after a site-directed cysteine substitution was made in the second outermost Arg in each S4 (DI-R2C and DII-R2C). We first determined the fraction of gating charge contributed by the individual S4's to maximal gating current (Qmax), and found that the outermost Arg residue in each S4 contributed ∼19% to Qmax with minimal contributions by other arginines. Stabilization of the S4's in DI-R2C and DII-R2C was confirmed by measuring the expected reduction in Qmax. In DI-R2C, stabilization resulted in a decrease in peak INa of ∼45%, while its peak current-voltage (I-V) and voltage-dependent Na channel availability (SSI) curves were nearly unchanged from wild type (WT). In contrast, stabilization of the DII-R2C enhanced activation with a negative shift in the peak I-V relationship by -7 mV and a larger -17 mV shift in the voltage-dependent SSI curve. Furthermore, its INa decay time constants and time-to-peak INa became more rapid than WT. An explanation for these results is that the depolarized conformation of DII-S4, but not DI-S4, affects the receptor for the inactivation particle formed by the interdomain linker between DIII and IV. In addition, the leftward shifts of both activation and inactivation and the decrease in Gmax after stabilization of the DII-S4 support previous studies that showed ß-scorpion toxins trap the voltage sensor of DII in an activated conformation.

A molecular model of the inner pore of the Ca channel in its open state.

Structure of the Ca channel open pore is unlikely to be the same as that of the K channel because Ca channels do not contain the hinge residues Gly or Pro. The Ca channel does not have a wide entry into the inner pore, as is found in K channels. First we sought to simulate the open state of the Ca channel by modeling forced opening of the KcsA channel using a procedure of restrained minimization with distance constraints at the level of the α-helical bundle, corresponding to segments Thr-107-Val-115. This produced an intermediate open state, which was populated by amino acid residues of Ca channels and then successively optimized until the opening of the pore reached a diameter of about 10 Å, large enough to allow verapamil to enter and block the Ca channel from inside. Although this approach produced a sterically plausible structure, it was in significant disagreement with the MTSET accessibility data for single cysteine mutations of S6 segments of the P/Q channel(1) that do not fit with an α-helical pattern. Last we explored the idea that the four S6 segments of Ca channels may contain intra-molecular deformations that lead to reorientation of its side chains. After introduction of π-bulges, the model agreed with the MTSET accessibility data. MTSET modification of a cysteine at the C-end of only one S6 could produce physical occlusion and block of the inner pore of the open Ca channel, as observed experimentally, and as expected if the pore opening is narrower than that of K channels.

The N terminus of connexin37 contains an alpha-helix that is required for channel function.

The cytoplasmic N-terminal domain of connexins has been implicated in multiple aspects of gap junction function, including connexin trafficking/assembly and channel gating. A synthetic peptide corresponding to the first 23 amino acids of human connexin37 was prepared, and circular dichroism and nuclear magnetic resonance studies showed that this N-terminal peptide was predominantly alpha-helical between glycine 5 and glutamate 16. The importance of this structure for localization of the protein at appositional membranes and channel function was tested by expression of site-directed mutants of connexin37 in which amino acids leucine 10 and glutamine 15 were replaced with prolines or alanines. Wild type connexin37 and both substitution mutants localized to appositional membranes between transfected HeLa cells. The proline mutant did not allow intercellular transfer of microinjected neurobiotin; the alanine mutant allowed transfer, but less extensively than wild type connexin37. When expressed alone in Xenopus oocytes, wild type connexin37 produced hemichannel currents, but neither of the double substitution mutants produced detectable currents. The proline mutant (but not the alanine mutant) inhibited co-expressed wild type connexin37. Taken together, our data suggest that the alpha-helical structure of the connexin37 N terminus may be dispensable for protein localization, but it is required for channel and hemichannel function.

Outward stabilization of the S4 segments in domains III and IV enhances lidocaine block of sodium channels.

The anti-arrhythmic drug lidocaine has been shown to have a lower affinity for block of voltage-gated sodium channels at hyperpolarized potentials compared to depolarized potentials. Concomitantly, lidocaine reduces maximum gating charge (Qmax) by 40% resulting from the complete stabilization of the S4 in domain III in an outward, depolarized position and partial stabilization of the S4 in domain IV in wild-type Na+ channels (Na(V)1.5). To investigate whether the pre-positioning of the S4 segments in these two domains in a depolarized conformation increases affinity for lidocaine block, a cysteine residue was substituted for the 3rd outermost charged residue in the S4 of domain III (R3C-DIII) and for the 2nd outermost Arg in S4 of domain IV (R2C-DIV) in Na(V)1.5. After biotinylation by exposure to extracellular MTSEA-biotin the mutated S4s became stabilized in an outward, depolarized position. For Na+ channels containing both mutations (R3C-DIII + R2C-DIV) the IC50 for rested-state lidocaine block decreased from 194 +/- 15 microM in control to 28 +/- 2 microM after MTSEA-biotin modification. To determine whether an intact inactivation gate (formed by the linker between domains III and IV) was required for local anaesthetic drugs to modify Na+ channel gating currents, a Cys was substituted for the Phe in the IFM motif of the inactivation gate (ICM) and then modified by intracellular MTSET (WT-ICM(MTSET)) before exposure to intracellular QX-222, a quarternary amine. Although WT-ICM(MTSET) required higher concentrations of drug to block I(Na) compared to WT, Qmax decreased by 35% and the V1/2 shifted leftward as previously demonstrated for WT. The effect of stabilization of the S4s in domains III and IV in the absence of an intact inactivation gate on lidocaine block was determined for R3C-DIII + ICM, R2C-DIV + ICM and R3C-DIII + R2C-DIV + ICM, and compared to WT-ICM. IC50 values were 1360 +/- 430 microM, 890 +/- 70 microM, 670 +/- 30 microM and 1920 +/- 60 microM, respectively. Thermodynamic mutant-cycle analysis was consistent with additive (i.e. independent) contributions from stabilization of the individual S4s in R3C-DIII + ICM and R2C-DIV + ICM. We conclude that the positions of the S4s in domains III and IV are major determinants of the voltage dependence of lidocaine affinity.

Charge at the lidocaine binding site residue Phe-1759 affects permeation in human cardiac voltage-gated sodium channels.

Our homology molecular model of the open/inactivated state of the Na(+) channel pore predicts, based on extensive mutagenesis data, that the local anaesthetic lidocaine docks eccentrically below the selectivity filter, such that physical occlusion is incomplete. Electrostatic field calculations suggest that the drug's positively charged amine produces an electrostatic barrier to permeation. To test the effect of charge at this pore level on permeation in hNa(V)1.5 we replaced Phe-1759 of domain IVS6, the putative binding site for lidocaine's alkylamino end, with positively and negatively charged residues as well as the neutral cysteine and alanine. These mutations eliminated use-dependent lidocaine block with no effect on tonic/rested state block. Mutant whole cell currents were kinetically similar to wild type (WT). Single channel conductance (gamma) was reduced from WT in both F1759K (by 38%) and F1759R (by 18%). The negatively charged mutant F1759E increased gamma by 14%, as expected if the charge effect were electrostatic, although F1759D was like WT. None of the charged mutations affected Na(+)/K(+) selectivity. Calculation of difference electrostatic fields in the pore model predicted that lidocaine produced the largest positive electrostatic barrier, followed by lysine and arginine, respectively. Negatively charged glutamate and aspartate both lowered the barrier, with glutamate being more effective. Experimental data were in rank order agreement with the predicted changes in the energy profile. These results demonstrate that permeation rate is sensitive to the inner pore electrostatic field, and they are consistent with creation of an electrostatic barrier to ion permeation by lidocaine's charge.

Site-3 toxins and cardiac sodium channels.

Site-3 toxins are small polypeptide venoms from scorpions, sea anemones, and spiders that bind with a high specificity to the extracellular surface of voltage-gated Na channels. After binding to a site near the S4 segment in domain IV the toxin causes disruption of the normal fast inactivation transition resulting in a marked prolongation of the action potentials of excitable tissues including those of cardiac and skeletal muscle and nerve. In this review we discuss the specific binding interactions between residues of the toxin and those of the Na channel, and the specific modification of Na channel kinetic behavior leading to a change in fast inactivation focusing on interactions deduced primarily from the study of sea anemone toxins and the cardiac Na channel (Na(V)1.5). We also illustrate the usefulness of site-3 toxins in the study of altered Na channel behavior by drug-modification.

An inner pore residue (Asn406) in the Nav1.5 channel controls slow inactivation and enhances mibefradil block to T-type Ca2+ channel levels.

Mibefradil is a tetralol derivative once marketed to treat hyper-tension. Its primary target is the T-type Ca(2+) channel (IC(50), approximately 0.1-0.2 microM), but it also blocks Na(+),K(+),Cl(-), and other Ca(2+) channels at higher concentrations. We have recently reported state-dependent mibefradil block of Na(+) channels in which apparent affinity was enhanced when channels were recruited to slow-inactivated conformations. The structural determinants controlling mibefradil block have not been identified, although evidence suggests involvement of regions near or within the inner pore. We tested whether mibefradil interacts with the local anesthetic (LA) binding site, which includes residues in the S6 segments of domains (D) I, III, and IV. Mutagenesis of DIII S6 and DIVS6 did not reveal critical binding determinants. Substitution of Asn406 in DI S6 of cardiac Na(v)1.5, however, altered affinity in a manner dependent on the identity of the substituting residue. Replacing Asn406 with a phenylalanine or a cysteine increased affinity by 4- and 7-fold, respectively, thus conferring T-type Ca(2+) channel-like mibefradil sensitivity to the Na(+) channel. A series of other substitutions that varied in size, charge, and hydrophobicity had minimal effects on mibefradil block, but all mutations dramatically altered the magnitude and voltage-dependence of slow inactivation, consistent with data in other isoforms. Channels did not slow-inactivate, however, at the voltages used to assay mibefradil block, supporting the idea that Asn406 lies within or near the mibefradil binding site.

Role of Domain IV/S4 outermost arginines in gating of T-type calcium channels.

The role of the outermost three charged residues of Domain IV/S4 in controlling gating of Ca(v)3.2 was investigated using single substitutions of each arginine with glutamine, cysteine, histidine, and lysine in a Flp-In-293 cell line, in which expression levels could be compared. Channel density, based on gating charge measurements, was ~125,000 channels/cell (10 fC/pF), except for R2Q and R3C, which expressed at lower levels. Channels substituted at Arg-1715 (R1C, R1Q, R1H) demonstrated such modest changes that a role in voltage sensing could not be determined. Arg-1718 (R2) made a contribution to activation voltage sensing, and the channel was sensitive to the geometry of side-chain substitutions at this position. Arg-1721 (R3) substitutions produced complex kinetic changes that together suggested that geometry made a larger contribution than charge. Current decay at positive potentials (O-->I) exponentially approached a constant value for all mutants except R2K channels, which were biphasically dependent on potential. R2K channels also displayed slowed deactivation with reduced voltage dependence despite near control values for conductance. Voltage-dependent accessibility of R to C mutants, evaluated with intracellularly and extracellularly applied methanthiosulfonate (MTS) reagents, showed that both R2 and R3 were exposed only when cells were depolarized, although it was not necessary for channels to open. Together, the data indicate that Domain IV/S4 is an activation domain and is not involved in inactivation from the open state.

Charge immobilization of the voltage sensor in domain IV is independent of sodium current inactivation.

Recovery from fast inactivation in voltage-dependent Na+ channels is associated with a slow component in the time course of gating charge during repolarization (i.e. charge immobilization), which results from the slow movement of the S4 segments in domains III and IV (S4-DIII and S4-DIV). Previous studies have shown that the non-specific removal of fast inactivation by the proteolytic enzyme pronase eliminated charge immobilization, while the specific removal of fast inactivation (by intracellular MTSET modification of a cysteine substituted for the phenylalanine in the IFM motif, ICMMTSET, in the inactivation particle formed by the linker between domains III and IV) only reduced the amount of charge immobilization by nearly one-half. To investigate the molecular origin of the remaining slow component of charge immobilization we studied the human cardiac Na+ channel (hH1a) in which the outermost arginine in the S4-DIV, which contributes approximately 20% to total gating charge (Qmax), was mutated to a cysteine (R1C-DIV). Gating charge could be fully restored in R1C-DIV by exposure to extracellular MTSEA, a positively charged methanethiosulphonate reagent. The RIC-DIV mutation was combined with ICMMTSET to remove fast inactivation, and the gating currents of R1C-DIV-ICM(MTSET) were recorded before and after modification with MTSEAo. Prior to MTSEAo, the time course of the gating charge during repolarization (off-charge) was best described by a single fast time constant. After MTSEA, the off-charge had both fast and slow components, with the slow component accounting for nearly 35% of Qmax. These results demonstrate that the slow movement of the S4-DIV during repolarization is not dependent upon the normal binding of the inactivation particle.

Accessibility of mid-segment domain IV S6 residues of the voltage-gated Na+ channel to methanethiosulfonate reagents.

The inner pore of the voltage-gated Na+ channel is predicted by the structure of bacterial potassium channels to be lined with the four S6 alpha-helical segments. Our previously published model of the closed pore based on the KcsA structure, and our new model of the open pore based on the MthK structure predict which residues in the mid-portion of S6 face the pore. We produced cysteine mutants of the mid-portion of domain IV-S6 (Ile-1575-Leu-1591) in NaV 1.4 and tested their accessibility to intracellularly and extracellularly placed positively charged methanethiosulfonate (MTS) reagents. We found that only two mutants, F1579C and V1583C, were accessible to both outside and inside 2-(aminoethyl)-methanethiosulfonate hydrobromide (MTSEA) Further study of those mutants showed that efficient closure of the fast inactivation gate prevented block by inside [2-(trimethylammonium)ethyl]methanethiosulfonate bromide (MTSET) at slow stimulation rates. When fast inactivation was inhibited by exposure to anthropleurin B (ApB), increasing channel open time, both mutants were blocked by inside MTSET at a rate that depended on the amount of time the channel was open. Consistent with the fast inactivation gate limiting access to the pore, in the absence of ApB, inside MTSET produced block when the cells were stimulated at 5 or 20 Hz. We therefore suggest that the middle of IV-S6 is an alpha-helix, and we propose a model of the open channel, based on MthK, in which Phe-1579 and Val-1583 face the pore.

Role of Asn-16 and Ser-19 in anthopleurin B binding. Implications for the electrostatic nature of Na(V) site 3.

Anthopleurin B (ApB) is a type 1 sea anemone toxin, which binds to voltage-sensitive sodium channels (Na(V)'s), thereby delaying channel inactivation. Previous work from our laboratories has demonstrated that the structurally unconstrained region involving residues 8-17 of this polypeptide, designated the Arg-14 loop, is important for full toxin affinity (Seibert et al., (2003) Biochemistry 42, 14515). Within this region, important contributions are made by residues Arg-12 and Leu-18 (Gallagher and Blumenthal, (1994) J. Biol. Chem. 269, 254; Dias-Kadambi et al., (1996) J. Biol. Chem. 271, 23828). Moreover, replacement of glycine residues found at positions 10 or 15 of the loop by alanine has been shown to have profound, isoform-selective effects on toxin-binding kinetics (Seibert et al., (2003)Biochemistry 42, 14515). To thoroughly understand the importance of this entire region, the work described here investigates the contribution of ApB residues Asn-16, Thr-17, and Ser-19 to toxin affinity and isoform selectivity. Our results demonstrate that residues within and proximal to the C terminus of the Arg-14 loop are important modulators of ApB affinity for Na(V) channels, indicating that the loop and channel site 3 are likely in close contact. A comparison of the effects of multiple replacements at each position reveals that Asn-16 and Ser-19 are involved in binding, whereas Thr-17 is not. The fact that anionic replacements for Asn-16 or Ser-19 are highly deleterious for toxin binding strongly suggests that site 3 contains either formal anionic residues or regions of high electron density, which could be formed by aromatic clusters. These data represent the first indication of the presence of such residues or regions within Na(V) site 3.

Arg-14 loop of site 3 anemone toxins: effects of glycine replacement on toxin affinity.

Anthopleurin B (ApB) is a high-affinity sea anemone neurotoxin that interacts with voltage-sensitive sodium (Na(V)) channels, causing a delay in channel inactivation. The solution structures of all known anemone toxins having this activity include a poorly defined region encompassing ApB residues 8-17, which we call the Arg-14 loop. We propose that the inherent mobility of the Arg-14 loop is necessary for the toxins' ability to maintain a high-affinity channel complex throughout the continual conformational transitions experienced by the channel during its functional cycle. We have previously shown that Arg-12, located in this loop, and Leu-18, which is adjacent, are important for ApB activity. Here, we characterized the role of two glycines located within the loop (Gly-10 and Gly-15) and an additional glycine positioned immediately C-terminal to it (Gly-20). We used site-directed replacement by alanine to assess the functional contribution to toxin binding of each of these residues singly and in combination. Gly-20 was found to be an essential toxin folding determinant; Gly-10 and Gly-15 were important for determining toxin affinity. Compared to wild-type toxin, the G10A and G15A toxins displayed significantly higher K(D) values for both cardiac (Na(V)1.5) and neuronal (Na(V)1.2) channels, although both demonstrated greater isoform discrimination for Na(V)1.5 than did wild-type ApB. For both G10A and G15A, significant Na(V) isoform differences were evident for on- and off-rates, with the most dramatic effect of a single mutation being the 467-fold reduction in the on-rate for G10A binding to Na(V)1.2, suggestive of a more accommodating binding site on Na(V)1.5 as compared to Na(V)1.2. Because alanine replacement of glycines is known to be associated with reduced backbone freedom, these results suggest an essential role for Arg-14 loop flexibility in toxin function, although a direct steric effect of the mutant methyl group cannot be excluded.

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 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.