Domain III S4 in closed-state fast inactivation: Insights from a periodic paralysis mutation

Heterologous expression of sodium channel mutations in hypokalemic periodic paralysis reveals 2 variants on channel dysfunction. Charge-reducing mutations of voltage sensing S4 arginine residues alter channel gating as typically studied with expression in mammalian cells. These mutations also produce leak currents through the voltage sensor module, as typically studied with expression in Xenopus oocytes. DIIIS4 mutations at R3 in the skeletal muscle sodium channel produce gating defects and omega current consistent with the phenotype of reduced excitability. Here, we confirm DIIIS4 R3C gating defects in the oocyte expression system for fast inactivation and its recovery. We provide novel data for the effects of the cysteine mutation on voltage sensor movement, to further our understanding of sodium channel defects in hypokalemic periodic paralysis. Gating charge movement and its remobilization are selectively altered by the mutation at hyperpolarized membrane potential, as expected with reduced serum potassium.     

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.     

Gating of the bacterial sodium channel, NaChBac: voltage-dependent charge movement and gating currents

The bacterial sodium channel, NaChBac, from Bacillus halodurans provides an excellent model to study structure-function relationships of voltage-gated ion channels. It can be expressed in mammalian cells for functional studies as well as in bacterial cultures as starting material for protein purification for fine biochemical and biophysical studies. Macroscopic functional properties of NaChBac have been described previously (Ren, D., B. Navarro, H. Xu, L. Yue, Q. Shi, and D.E. Clapham. 2001. Science. 294:2372-2375). In this study, we report gating current properties of NaChBac expressed in COS-1 cells. Upon depolarization of the membrane, gating currents appeared as upward inflections preceding the ionic currents. Gating currents were detectable at -90 mV while holding at -150 mV. Charge-voltage (Q-V) curves showed sigmoidal dependence on voltage with gating charge saturating at -10 mV. Charge movement was shifted by -22 mV relative to the conductance-voltage curve, indicating the presence of more than one closed state. Consistent with this was the Cole-Moore shift of 533 micros observed for a change in preconditioning voltage from -160 to -80 mV. The total gating charge was estimated to be 16 elementary charges per channel. Charge immobilization caused by prolonged depolarization was also observed; Q-V curves were shifted by approximately -60 mV to hyperpolarized potentials when cells were held at 0 mV. The kinetic properties of NaChBac were simulated by simultaneous fit of sodium currents at various voltages to a sequential kinetic model. Gating current kinetics predicted from ionic current experiments resembled the experimental data, indicating that gating currents are coupled to activation of NaChBac and confirming the assertion that this channel undergoes several transitions between closed states before channel opening. The results indicate that NaChBac has several closed states with voltage-dependent transitions between them realized by translocation of gating charge that causes activation of the channel.     

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

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

Fast and slow voltage sensor movements in HERG potassium channels

HERG encodes an inwardly-rectifying potassium channel that plays an important role in repolarization of the cardiac action potential. Inward rectification of HERG channels results from rapid and voltage-dependent inactivation gating, combined with very slow activation gating. We asked whether the voltage sensor is implicated in the unusual properties of HERG gating: does the voltage sensor move slowly to account for slow activation and deactivation, or could the voltage sensor move rapidly to account for the rapid kinetics and intrinsic voltage dependence of inactivation? To probe voltage sensor movement, we used a fluorescence technique to examine conformational changes near the positively charged S4 region. Fluorescent probes attached to three different residues on the NH₂-terminal end of the S4 region (E518C, E519C, and L520C) reported both fast and slow voltage-dependent changes in fluorescence. The slow changes in fluorescence correlated strongly with activation gating, suggesting that the slow activation gating of HERG results from slow voltage sensor movement. The fast changes in fluorescence showed voltage dependence and kinetics similar to inactivation gating, though these fluorescence signals were not affected by external tetraethylammonium blockade or mutations that alter inactivation. A working model with two types of voltage sensor movement is proposed as a framework for understanding HERG channel gating and the fluorescence signals.     

Central Charged Residues in DIIIS4 Regulate Deactivation Gating in Skeletal Muscle Sodium Channels

Summary 1. Mutations in the S4 segment of domain III in the voltage gated skeletal muscle sodium channel hNa_V1.4 were constructed to test the roles of each charged residue in deactivation gating. Mutations comprised charge reversals at K1-R6, charge neutralization, and substitution at R4 and R5. 2. Charge-reversing mutations at R4 and R5 produced the greatest alteration of activation parameters compared to hNa_V1.4. Effects included depolarization of the conductance/voltage (g/V) curve, decreased valence and slowing of kinetics. 3. Reversal of charge at R2 to R4 hyperpolarized, and reversal at R5 or R6 depolarized the h _∞ curve. Most DIIIS4 mutations slowed inactivation from the open state. R4E slowed closed state fast inactivation and R5E inhibited its completion. 4. Deactivation from the open and/or inactivated state was prolonged in mutations reversing charge at R2 to R4 but accelerated by reversal of charge at R5 or R6. Effects were most pronounced at central charges R4 and R5. 5. Charge and structure each contribute to effects of mutations at R4 and R5 on channel gating. Effects of mutations on activation and deactivation at R4 and, to a lesser extent R5, were primarily owing to charge alteration, whereas effects on fast inactivation were charge independent.     

Built for speed

Many of us were taught in high school biology that the action potential waveform in nerves and other excitable tissues was generated by an initial rapid influx of external Na⁺ ions across the plasma membrane, followed by an outward movement of intracellular K⁺ ions. The former event, mediated by voltage-gated Na⁺ channels, is responsible for the fast depolarizing upstroke of the action potential, while voltage-gated K+ channels are responsible for the subsequent repolarizing phase, which largely controls action potential duration. Although Hodgkin and Huxley described the fundamental importance of this sequential activation process more than 60 y ago, the molecular and structural details underlying the faster activation of voltage-gated Na⁺ (Nav) vs. K⁺ (Kv) channels have yet to be fully resolved.     

Histidine scanning mutagenesis of basic residues of the S4 segment of the shaker k+ channel

The voltage sensor of the Shaker potassium channel is comprised mostly of positively charged residues in the putative fourth transmembrane segment, S4 (Aggarwal, S.K., and R. MacKinnon. 1996. Neuron. 16:1169-1177; Seoh, S.-A., D. Sigg, D.M. Papazian, and F. Bezanilla. 1996. Neuron. 16:1159-1167). Movement of the voltage sensor in response to a change in the membrane potential was examined indirectly by measuring how the accessibilities of residues in and around the sensor change with voltage. Each basic residue in the S4 segment was individually replaced with a histidine. If the histidine tag is part of the voltage sensor, then the gating charge displaced by the voltage sensor will include the histidine charge. Accessibility of the histidine to the bulk solution was therefore monitored as pH-dependent changes in the gating currents evoked by membrane potential pulses. Histidine scanning mutagenesis has several advantages over other similar techniques. Since histidine accessibility is detected by labeling with solution protons, very confined local environments can be resolved and labeling introduces minimal interference of voltage sensor motion. After histidine replacement of either residue K374 or R377, there was no titration of the gating currents with internal or external pH, indicating that these residues do not move in the transmembrane electric field or that they are always inaccessible. Histidine replacement of residues R365, R368, and R371, on the other hand, showed that each of these residues traverses entirely from internal exposure at hyperpolarized potentials to external exposure at depolarized potentials. This translocation enables the histidine to transport protons across the membrane in the presence of a pH gradient. In the case of 371H, depolarization drives the histidine to a position that forms a proton pore. Kinetic models of titrateable voltage sensors that account for proton transport and conduction are presented. Finally, the results presented here are incorporated into existing information to propose a model of voltage sensor movement and structure.     

State-dependent block of wild-type and inactivation-deficient Na+ channels by flecainide

The antiarrhythmic agent flecainide appears beneficial for painful congenital myotonia and LQT-3/DeltaKPQ syndrome. Both diseases manifest small but persistent late Na+ currents in skeletal or cardiac myocytes. Flecainide may therefore block late Na+ currents for its efficacy. To investigate this possibility, we characterized state-dependent block of flecainide in wild-type and inactivation-deficient rNav1.4 muscle Na+ channels (L435W/L437C/A438W) expressed with beta1 subunits in Hek293t cells. The flecainide-resting block at -140 mV was weak for wild-type Na+ channels, with an estimated 50% inhibitory concentration (IC50) of 365 micro M when the cell was not stimulated for 1,000 s. At 100 micro M flecainide, brief monitoring pulses of +30 mV applied at frequencies as low as 1 per 60 s, however, produced an approximately 70% use-dependent block of peak Na+ currents. Recovery from this use-dependent block followed an exponential function, with a time constant over 225 s at -140 mV. Inactivated wild-type Na+ channels interacted with flecainide also slowly at -50 mV, with a time constant of 7.9 s. In contrast, flecainide blocked the open state of inactivation-deficient Na+ channels potently as revealed by its rapid time-dependent block of late Na+ currents. The IC50 for flecainide open-channel block at +30 mV was 0.61 micro M, right within the therapeutic plasma concentration range; on-rate and off-rate constants were 14.9 micro M-1s-1 and 12.2 s-1, respectively. Upon repolarization to -140 mV, flecainide block of inactivation-deficient Na+ channels recovered, with a time constant of 11.2 s, which was approximately 20-fold faster than that of wild-type counterparts. We conclude that flecainide directly blocks persistent late Na+ currents with a high affinity. The fast-inactivation gate, probably via its S6 docking site, may further stabilize the flecainide-receptor complex in wild-type Na+ channels.     

Sodium currents in the giant axon of the crabCarcinus maenas

Summary Measurements were made of the kinetics and steady-state properties of the sodium conductance changes in the giant axon of the crabCarcinus maenas. The conductance measurements were made in the presence of small concentrations of tetrodotoxin and as much electrical compensation as possible in order to minimize errors caused by the series resistance. After an initial delay of 10–150 sec, the conductance increase during depolarizing voltage clamp pulses followed the Hodgkin-Huxley kinetics. Values of the time constant for the activation of the sodium conductance lay on a bell-shaped curve with a maximum under 180 sec at –40 mV (at 18°C). Values of the time constant for the inactivation of the sodium conductance were also fitted using a bell-shaped curve with a maximum under 7 msec at –70 mV. The effects of membrane potential on the fraction of Na channels available for activation studied using double pulse protocols suggest that hyperpolarizing potentials more negative than –100 mV lock a fraction of the Na channels in a closed conformation.     

Mutations of SCN4A gene cause different diseases: 2 case reports and literature review

SCN4A encodes the Nav1.4 channel and mutations in SCN4A lead to different ionic channelopathies. In this study, one sporadic individual of periodic paralysis, one paramyotonia family and 200 normal healthy controls are enrolled. Genomic DNA was extracted from peripheral blood leukocytes, followed by polymerase chain reaction and DNA sequencing of candidate genes, including SCN4A and CACNA1S. As a result, heterozygous mutations c.2024G>A (R675Q) and c.1333G>A (V445M) of gene SCN4A were identified in the hypokalemic periodic paralysis patient and the paramyotonia congenita family respectively. Both mutations were not detected in healthy controls. Compared with reported cases, patients with mutation R675Q usually do not present hypokalemic periodic paralysis but hyperkalemic or normokalemic periodic paralysis. The mutation V445M was first reported in Chinese patients with nondystrophic myotonias. In addition, we carried out literature review by summarizing clinical features of the 2 mutations and establish the genotype–phenotype correlations to provide guidance for diagnosis.     

Block of inactivation-deficient Na+ channels by local anesthetics in stably transfected mammalian cells: evidence for drug binding along the activation pathway

According to the classic modulated receptor hypothesis, local anesthetics (LAs) such as benzocaine and lidocaine bind preferentially to fast-inactivated Na(+) channels with higher affinities. However, an alternative view suggests that activation of Na(+) channels plays a crucial role in promoting high-affinity LA binding and that fast inactivation per se is not a prerequisite for LA preferential binding. We investigated the role of activation in LA action in inactivation-deficient rat muscle Na(+) channels (rNav1.4-L435W/L437C/A438W) expressed in stably transfected Hek293 cells. The 50% inhibitory concentrations (IC(50)) for the open-channel block at +30 mV by lidocaine and benzocaine were 20.9 +/- 3.3 microM (n = 5) and 81.7 +/- 10.6 microM (n = 5), respectively; both were comparable to inactivated-channel affinities. In comparison, IC(50) values for resting-channel block at -140 mV were >12-fold higher than those for open-channel block. With 300 microM benzocaine, rapid time-dependent block (tau approximately 0.8 ms) of inactivation-deficient Na(+) currents occurred at +30 mV, but such a rapid time-dependent block was not evident at -30 mV. The peak current at -30 mV, however, was reduced more severely than that at +30 mV. This phenomenon suggested that the LA block of intermediate closed states took place notably when channel activation was slow. Such closed-channel block also readily accounted for the LA-induced hyperpolarizing shift in the conventional steady-state inactivation measurement. Our data together illustrate that the Na(+) channel activation pathway, including most, if not all, transient intermediate closed states and the final open state, promotes high-affinity LA binding.     

Fast Inactivation in Shaker K+ Channels : Properties of Ionic and Gating Currents

Fast inactivating Shaker H4 potassium channels and nonconducting pore mutant Shaker H4 W434F channels have been used to correlate the installation and recovery of the fast inactivation of ionic current with changes in the kinetics of gating current known as “charge immobilization” (Armstrong, C.M., and F. Bezanilla. 1977. J. Gen. Physiol. 70:567–590.). Shaker H4 W434F gating currents are very similar to those of the conducting clone recorded in potassium-free solutions. This mutant channel allows the recording of the total gating charge return, even when returning from potentials that would largely inactivate conducting channels. As the depolarizing potential increased, the OFF gating currents decay phase at −90 mV return potential changed from a single fast component to at least two components, the slower requiring ∼200 ms for a full charge return. The charge immobilization onset and the ionic current decay have an identical time course. The recoveries of gating current (Shaker H4 W434F) and ionic current (Shaker H4) in 2 mM external potassium have at least two components. Both recoveries are similar at −120 and −90 mV. In contrast, at higher potentials (−70 and −50 mV), the gating charge recovers significantly more slowly than the ionic current. A model with a single inactivated state cannot account for all our data, which strongly support the existence of “parallel” inactivated states. In this model, a fraction of the charge can be recovered upon repolarization while the channel pore is occupied by the NH₂-terminus region.     

Differential blockage of two types of potassium channels in the crab giant axon

Summary Measurements were made of the kinetic and steady-state characteristics of the potassium conductance in the giant axon of the crabsCarcinus maenas andCancer pagirus. The conductance increase during depolarizing voltage-clamp pulses was analyzed assuming that two separate types of potassium channels exist in these axons (M. E. Quinta-Ferreira, E. Rojas and N. Arispe,J. Membrane Biol. 66:171–181, 1982). It is shown here that, with small concentrations of conventional K⁺-channel blockers, it is possible to differentially inhibit these channels. The potassium channels with activation and fast inactivation gating (m³h, Hodgkin-Huxley kinetics) were blocked by external application of 4 amino-pyridine (4-AP). The potassium channels with standard gating (n⁴, Hodgkin-Huxley kinetics) were preferentially inhibited by externally applied tetraethylammonium (TEA). The differential blockage of the two types of potassium conductance changes suggests that they represent two different populations of potassium channels.It is further shown here that blocking the early transient conductance increase leads to the inhibition of the repetitive electrical activity induced by constant depolarizing current injection in fibers fromCardisoma guanhumi.     

Gating currents of sodium channels in neurons of the rat trigeminal ganglia

Using a voltage-clamp technique and intracellular dialysis, gating currents of sodium channels were first recorded and studied in neurons of the rat trigeminal ganglia. The rising phase of gating currents lasted 30 to 70 µsec; these currents decayed in a monoexponential manner with a time constant equal to that for activation of the sodium current. Voltage dependences for the gating charge and sodium conductance were also nearly identical. Analysis of the activation of sodium conductance demonstrated that the power n of the activation variable in the equation used changed from more than 6 to 3 at test potentials of –30 mV and 0 mV, respectively. It is hypothesized that, with a change in the test potential within this voltage range, the cooperativity of activation undergoes a twofold decrease. In the presence of 2 mM caffeine or theophylline in the external solution, curves of the voltage dependence of the gating charge and sodium conductance shifted toward more negative values of the test potential, by 5.4 ± 0.7 mV, the maximum gating charge increased by 8.4 ± 3.2%, and the slope factor for both curves decreased by 9.2 ± 3.4%. Since the above effects were identical for both xanthines and developed under conditions of constant intracellular dialysis, i.e., under conditions where the effect of a change in the intracellular calcium concentration was ruled out, the most probable reason for these effects is a direct action of the tested agents on sodium ion channels, which facilitates the movement of gating charges.Neirofiziologiya/Neurophysiology, Vol. 36, Nos. 5/6, pp. 370–376, September–December, 2004.This revised version was published online in April 2005 with a corrected cover date and copyright year.     

N type rapid inactivation in human Kv1.4 channels: functional role of a putative C-terminal helix

Voltage gated potassium channels are tetrameric membrane proteins, which have a central role in cellular excitability. Human Kv1.4 channels open on membrane depolarization and inactivate rapidly by a ‘ball and chain’ mechanism whose molecular determinants have been mapped to the cytoplasmic N terminus of the channel. Here we show that the other terminal end of the channel also plays a role in channel inactivation. Swapping the C-terminal residues of hKv1.4 with those from two non-inactivating channels (hKv1.1 and hKv1.2) affects the rates of inactivation, as well as the recovery of the channel from the inactivated state. Secondary structure predictions of the hKv1.4 sequence reveal a helical structure at its distal C-terminal. Complete removal or partial disruption of this helical region results in channels with remarkably slowed inactivation kinetics. The ionic selectivity and voltage-dependence of channel opening were similar to hKv1.4, indicative of an unperturbed channel pore. These results demonstrate that fast inactivation is modulated by structural elements in the C-terminus, suggesting that the process involves the concerted action of the N- and C-termini.