S4 Segment Of Domain Research Articles

Novel idiopathic DCM-related SCN5A variants localised in DI-S4 predispose electrical disorders by reducing peak sodium current density

BackgroundVariants of SCN5A, encoding cardiac sodium channel, have been linked to the development of dilated cardiomyopathy (DCM). We aimed to explore novel SCN5A variants in patients with idiopathic DCM (iDCM)...

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 β-scorpion toxins trap the voltage sensor of DII in an activated conformation.

Lidocaine partially depolarizes the S4 segment in domain IV of the sodium channel

Previous studies have shown that lidocaine and other local anesthetic drugs (LAs) cause use-dependent block of sodium current (I (Na)), i.e., block that increases with membrane depolarization by allosteric coupling between drug binding in the inner pore and the S4s in domains III and IV. MTSET protection experiments have established that LAs stabilize DIIIS4 in an outward, depolarized position. Similar tests have not been reported for the DIVS4, although LAs have been shown to reduce DIV's contribution to total gating charge by about one third and to alter its movement such that it contributes more gating charge at negative potentials around -100mV compared to non-drug-bound sodium (Na) channels. To investigate whether lidocaine reduces the gating charge of DIVS4 by causing it to adopt either a depolarized position at rest or by restricting its outward movement upon depolarization, we performed MTSET protection experiments on I (Na) of the mutant Na channel, R1628C (R3C-DIV), in the presence and absence of 10mM lidocaine. The results indicate that lidocaine causes the DIVS4 to assume a more depolarized position, which facilitates its movement upon depolarization leading to the excess gating charge at potentials near -100mV.

Mutations in skeletal muscle

Mutations in skeletal muscle sodium channels (NaV1.4) cause periodic paralysis. Paramyotonia congenita and hyperkalemic periodic paralysis are caused by gain-of-function mutations spread widely through the protein, which increase channel activity and lead to repetitive firing or depolarization block. In contrast, mutations that cause hypokalemic (HypoPP) and normokalemic (NormoPP) periodic paralysis are localized in the outermost three gating-charge-carrying arginine residues (R1-R3) in the S4 segment in domain II, and they do not have major effects on sodium channel function as typically measured. Site-directed mutations of these residues cause gating pore current, a voltage-gated leak current through the voltage sensor (Sokolov et al., 2005); mutations of R1 and R2 cause gating pore current in the resting state, whereas mutation of R3 causes gating pore current in the activated state. Similar studies of the HypoPP mutant R2G revealed gating pore current of approximately 1% of peak current at the resting membrane potential, which was decreased by depolarization (Sokolov et al., 2007). This gating pore current was selective for Cs>K>Na and blocked by mM concentrations of divalent cations, Zn>Ba>Ca. A gating pore current of similar size was observed in the resting state for the HypoPP mutants R1H and R2H, but this current is selective for protons. In contrast to HypoPP, the mutations that cause NormoPP are in R3 (R3G/Q/W). All of these mutations cause gating pore conductance for sodium in the activated and slow-inactivated states, in which the voltage sensors are in their outward position. The common pathogenic feature of these mutations is likely to be depolarization and sodium overload, which are observed in patient biopsies. Dominant gain-of-function pathogenic effects may arise directly from excess sodium entry for R2G and R3G/Q/W and indirectly from excessive Na-H exchange for R1H and R2H.

The effect of the outermost basic residues in the S4 segments of the CaV3.1 T-type calcium channel on channel gating

The contribution of voltage-sensing S4 segments in domains I to IV of the T-type Ca(V)3.1 calcium channel to channel gating was investigated by the replacement of the uppermost charged arginine residues by neutral cysteines. In each construct, either a single (R180C, R834C, R1379C or R1717C) or a double (two adjacent domains) mutation was introduced. We found that the neutralisation of the uppermost arginines in the IS4, IIS4 and IIIS4 segments shifted the voltage dependence of channel activation in a hyperpolarising direction, with the most prominent effect in the IS4 mutant. In contrast, the voltage dependence of channel inactivation was shifted towards more negative membrane potentials in all four single mutant channels, and these effects were more pronounced than the effects on channel activation. Recovery from inactivation was affected by the IS4 and IIIS4 mutations. In double mutants, the effects on channel inactivation and recovery from inactivation, but not on channel activation, were additive. Exposure of mutant channels to the reducing agent dithiothreitol did not alter channel properties. In summary, our data indicate that the S4 segments in all four domains of the Ca(V)3.1 calcium channels contribute to voltage sensing during channel inactivation, while only the S4 segments in domains I, II and III play such role in channel activation. Furthermore, the removal of the outermost basic amino acids from the IVS4 and IIIS4 and, to a lesser extent, from IS4 segments stabilised the open state of the channel, whereas neutralization from that of IIS4 destabilised it.

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.

The effects of S4 segments on the voltage-dependence of inactivation for Cav3.1 calcium channels

T-type calcium channels exhibit fast voltage-dependent inactivation, for which the underlying structure-function relationship still remains unclear. To investigate the roles of S4 segments in voltage-dependent inactivation of T-type calcium channels, we created S4 replacement chimeras between Cav3.1 calcium channels (fast voltage-dependent inactivation) and Cav1.2 calcium channels (little voltage-dependent inactivation) by replacing S4s in Cav3.1 with the corresponding regions in Cav1.2. Wild type and chimeric channels were expressed in Xenopus oocytes and channel currents were recorded with two-electrode voltage-clamp. We showed that replacing S4 region in domain I shifted voltage-dependence for inactivation of Cav3.1 to the left, and the V 0.5 inact and k inact value were significantly changed. However replacing S4s in domains II–IV had no effects on the voltage-dependent in-activation properties. These results suggest that the roles of S4 segments in domains I–IV are different, and S4 in domain I is likely to be involved in voltage-dependent inactivation process. Its movement during membrane depolarization may trigger a conformational change in the inactivation gate.

Painful Channels

Paroxysmal extreme pain disorder (PEPD), previously known as familial rectal pain (FRP, OMIM 167400), is an inherited disease causing intense burning rectal, ocular, and submandibular pain and flushing. Fertleman et al. (this issue of Neuron) show that mutations in SCN9A, the gene encoding the sodium channel Na(V)1.7 channels, are responsible for this syndrome. Together with earlier work implicating a distinct class of functional mutations in SCN9A in a distinct inherited pain syndrome, these results point to Na(V)1.7 channels as key players in signaling nociceptive information and as a potential target for drug therapy of chronic pain.

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 V1.5). We also illustrate the usefulness of site-3 toxins in the study of altered Na channel behavior by drug-modification.

Molecular regions underlying the activation of low- and high-voltage activating calcium channels

We have studied two aspects of calcium channel activation. First, we investigated the molecular regions that are important in determining differences in activation between low- and high-voltage activated channels. For this, we made chimeras between the low-voltage activating Ca(V)3.1 channel and the high-voltage activating Ca(V)1.2 channel. Chimeras were expressed in oocytes, and calcium channel currents recorded by voltage clamp. For domain I, we found that the molecular region that is important in determining the voltage dependence of activation comprises the pore regions S5-P as well as P-S6, but surprisingly not the voltage sensor S1-S4 region, which might have been expected to play a major part. By contrast, the smaller, but still significant, modulating effects of domain II on activation properties were due to effects involving both S1-S4 and S5-S6 but not the I/II linker. Second, during channel activation we studied movement of the S4 segment in domain I of one of the chimeras, using cysteine-scanning mutagenesis. The reagent parachloromercuribenzensulfonate inhibited currents for mutants V263, A265, L266 and A268, but not for F269 and V271, and voltage dependence of inhibition for residue V263 indicated S4 movement, which occurred before channel opening. The data indicate movement outwards upon depolarisation so as to expose amino acids up to residue 268 in S4.

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.

Roles of Molecular Regions in Determining Differences between Voltage Dependence of Activation of CaV3.1 and CaV1.2 Calcium Channels

Voltage-dependent calcium channels are classified into low voltage-activated and high voltage-activated channels. We have investigated the molecular basis for this difference in voltage dependence of activation by constructing chimeras between a low voltage-activated channel (Ca(V)3.1) and a high voltage-activated channel (Ca(V)1.2), focusing on steady-state activation properties. Wild type and chimeras were expressed in oocytes, and two-electrode voltage clamp recordings were made of calcium channel currents. Replacement of domains I, III, or IV of the Ca 3.1 channel with the corresponding domain of Ca(V)1.2 led (V)to high voltage-activated channels; for these constructs the current/voltage (I/V) curves were similar to those for Ca(V)1.2 wild type. However, replacement of domain II gave only a small shift to the right of the I/V curve and modulation of the activation kinetics but did not lead to a high voltage-activating channel with an I/V curve like Ca 1.2. We also investigated the role of the voltage sensor (V)S4 by replacing the S4 segment of Ca(V)3.1 with that of Ca 1.2. For domain I, there was no shift in the I/V curve (V)as compared with Ca(V)3.1, and there were relatively small shifts to the right for domains III and IV. Taken together, these results suggest that domains I, III, and IV (rather than domain II) are apparently critical for channel opening and, therefore, contribute strongly to the difference in voltage dependence of activation between Ca 3.1 and Ca(V)1.2. However, the S4 segments in domains I, (V)III, and IV did not account for this difference in voltage dependence.

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 (Qmax) 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 Qmax 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.

From ionic currents to molecular mechanisms: the structure and function of voltage-gated sodium channels.

From ionic currents to molecular mechanisms: the structure and function of voltage-gated sodium channels.

Functional Consequences of Mutations in the Human α1ACalcium Channel Subunit Linked to Familial Hemiplegic Migraine

Mutations in alpha1A, the pore-forming subunit of P/Q-type calcium channels, are linked to several human diseases, including familial hemiplegic migraine (FHM). We introduced the four missense mutations linked to FHM into human alpha1A-2 subunits and investigated their functional consequences after expression in human embryonic kidney 293 cells. By combining single-channel and whole-cell patch-clamp recordings, we show that all four mutations affect both the biophysical properties and the density of functional channels. Mutation R192Q in the S4 segment of domain I increased the density of functional P/Q-type channels and their open probability. Mutation T666M in the pore loop of domain II decreased both the density of functional channels and their unitary conductance (from 20 to 11 pS). Mutations V714A and I1815L in the S6 segments of domains II and IV shifted the voltage range of activation toward more negative voltages, increased both the open probability and the rate of recovery from inactivation, and decreased the density of functional channels. Mutation V714A decreased the single-channel conductance to 16 pS. Strikingly, the reduction in single-channel conductance induced by mutations T666M and V714A was not observed in some patches or periods of activity, suggesting that the abnormal channel may switch on and off, perhaps depending on some unknown factor. Our data show that the FHM mutations can lead to both gain- and loss-of-function of human P/Q-type calcium channels.

Voltage Sensors in Domains III and IV, but Not I and II, Are Immobilized by Na + Channel Fast Inactivation

Using site-directed fluorescent labeling, we examined conformational changes in the S4 segment of each domain of the human skeletal muscle sodium channel (hSkM1). The fluorescence signals from S4 segments in domains I and II follow activation and are unaffected as fast inactivation settles. In contrast, the fluorescence signals from S4 segments in domains III and IV show kinetic components during activation and deactivation that correlate with fast inactivation and charge immobilization. These results indicate that in hSkM1, the S4 segments in domains III and IV are responsible for voltage-sensitive conformational changes linked to fast inactivation and are immobilized by fast inactivation, while the S4 segments in domains I and II are unaffected by fast inactivation.

Voltage Sensor–Trapping: Enhanced Activation of Sodium Channels by β-Scorpion Toxin Bound to the S3–S4 Loop in Domain II

Polypeptide neurotoxins alter ion channel gating by binding to extracellular receptor sites, even though the voltage sensors are in their S4 transmembrane segments. By analysis of sodium channel chimeras, a β-scorpion toxin is shown here to negatively shift voltage dependence of activation and enhance closed state inactivation by binding to a receptor site that requires glycine 845 (Gly-845) in the S3–S4 loop at the extracellular end of the S4 segment in domain II of the α subunit. Toxin action requires prior depolarization to drive the S4 voltage sensors outward, but these effects are lost in the mutant G845N. The results reveal a voltage sensor–trapping model of toxin action in which the IIS4 voltage sensor is trapped in its outward, activated position by toxin binding.

A de novo missense mutation of human cardiac Na + channel exhibiting novel molecular mechanisms of long QT syndrome

Mutations in a human cardiac Na + channel gene (SCN5A) are responsible for chromosome 3-linked congenital long QT syndrome (LQT3). Here we characterized a de novo missense mutation (R1623Q, S4 segment of domain 4) identified in an infant Japanese girl with a severe form of LQT3. When expressed in oocytes, mutant Na + channels exhibited only minor abnormalities in channel activation, but in contrast to three previously characterized LQT3 mutations, had significantly delayed macroscopic inactivation. Single channel analysis revealed that R1623Q channels have significantly prolonged open times with bursting behavior, suggesting a novel mechanism of pathophysiology in Na + channel-linked long QT syndrome.

Dihydropyridine receptor mutations cause hypokalemic periodic paralysis

Hypokalemic periodic paralysis (hypoKPP) is an autosomal dominant skeletal muscle disorder manifested by episodic weakness associated with low serum potassium. Genetic linkage analysis has localized the hypoKPP gene to chromosome 1q31–q32 near a dihydropyridine (DHP) receptor gene. This receptor functions as a voltage-gated calcium channel and is also critical for excitation-contraction coupling in a voltage-sensitive and calcium-independent manner. We have characterized patient-specific DHP receptor mutations in 11 probands of 33 independent hypoKPP kindreds that occur at one of two adjacent nucleotides within the same codon and predict substitution of a highly conserved arginine in the S4 segment of domain 4 with either histidine or glycine. In one kindred, the mutation arose de novo. Taken together, these data establish this DHP receptor as the hypoKPP gene. We are unaware of any other human diseases presently known to result from DHP receptor mutations.

Sodium channel mutations in paramyotonia congenita uncouple inactivation from activation

Mutations in the adult human skeletal muscle Na + channel a subunit cause the disease paramyotonia congenita. Two paramyotonia congenita mutations, R1448H and R1448C, substitute histidine and cysteine for arginine in the S4 segment of domain 4. These mutations, expressed in a cell line, have only small effects on the activation of Na+ currents, but mutant channels inactivate more slowly with less voltage dependence than wild-type channels and exhibit an enhanced rate of recovery from inactivation. Increase of extracellular pH made the rate of inactivation of RI 448H similar to that of R1448C, suggesting that this residue has an extracellular location and that its charge is important for normal inactivation. Analysis of single-channel data reveals that mutant channels inactivate normally from closed states, but poorly from the open state. The data suggest a critical role for the S4 helix of domain 4 in coupling between activation and inactivation.