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

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

A high-Na(+) conduction state during recovery from inactivation in the K(+) channel Kv1.5

Na(+) conductance through cloned K(+) channels has previously allowed characterization of inactivation and K(+) binding within the pore, and here we have used Na(+) permeation to study recovery from C-type inactivation in human Kv1.5 channels. Replacing K(+) in the solutions with Na(+) allows complete Kv1.5 inactivation and alters the recovery. The inactivated state is nonconducting for K(+) but has a Na(+) conductance of 13% of the open state. During recovery, inactivated channels progress to a higher Na(+) conductance state (R) in a voltage-dependent manner before deactivating to closed-inactivated states. Channels finally recover from inactivation in the closed configuration. In the R state channels can be reactivated and exhibit supernormal Na(+) currents with a slow biexponential inactivation. Results suggest two pathways for entry to the inactivated state and a pore conformation, perhaps with a higher Na(+) affinity than the open state. The rate of recovery from inactivation is modulated by Na(+)(o) such that 135 mM Na(+)(o) promotes the recovery to normal closed, rather than closed-inactivated states. A kinetic model of recovery that assumes a highly Na(+)-permeable state and deactivation to closed-inactivated and normal closed states at negative voltages can account for the results. Thus these data offer insight into how Kv1. 5 channels recover their resting conformation after inactivation and how ionic conditions can modify recovery rates and pathways.     

Constitutive inactivation of the hKv1.5 mutant channel, H463G, in K+-free solutions at physiological pH

Extracellular acidification and reduction of extracellular K⁺ are known to decrease the currents of some voltage-gated potassium channels. Although the macroscopic conductance of WT hKv1.5 channels is not very sensitive to [K⁺]_o at pH 7.4, it is very sensitive to [K⁺]_o at pH 6.4, and in the mutant, H463G, the removal of K⁺ _o virtually eliminates the current at pH 7.4. We investigated the mechanism of current regulation by K⁺ _o in the Kv1.5 H463G mutant channel at pH 7.4 and the wild-type channel at pH 6.4 by taking advantage of Na⁺ permeation through inactivated channels. Although the H463G currents were abolished in zero [K⁺]_o, robust Na⁺ tail currents through inactivated channels were observed. The appearnnce of H463G Na⁺ currents with a slow rising phase on repolarization after a very brief depolarization (2 ms) suggests that channels could activate directly from closed-inactivated states. In wild-type channels, when intracellular K⁺ was replaced by NMG⁺ and the inward Na⁺ current was recorded, addition of 1 mM K⁺ prevented inactivation, but changing pH from 7.4 to 6.4 reversed this action. The data support the idea that C-type inactivation mediated at R487 in Kv1.5 channels is influenced by H463 in the outer pore. We conclude that both acidification and reduction of [K⁺]_o inhibit Kv1.5 channels through a common mechananism (i.e., by increasing channel inactivation, which occurs in the resting state or develops very rapidly after activation).     

Voltage dependence of slow inactivation in Shaker potassium channels results from changes in relative K(+) and Na(+) permeabilities

Time constants of slow inactivation were investigated in NH(2)-terminal deleted Shaker potassium channels using macro-patch recordings from Xenopus oocytes. Slow inactivation is voltage insensitive in physiological solutions or in simple experimental solutions such as K(+)(o)//K(+)(i) or Na(+)(o)//K(+)(i). However, when [Na(+)](i) is increased while [K(+)](i) is reduced, voltage sensitivity appears in the slow inactivation rates at positive potentials. In such solutions, the I-V curves show a region of negative slope conductance between approximately 0 and +60 mV, with strongly increased outward current at more positive voltages, yielding an N-shaped curvature. These changes in peak outward currents are associated with marked changes in the dominant slow inactivation time constant from approximately 1.5 s at potentials less than approximately +60 mV to approximately 30 ms at more than +150 mV. Since slow inactivation in Shaker channels is extremely sensitive to the concentrations and species of permeant ions, more rapid entry into slow inactivated state(s) might indicate decreased K(+) permeation and increased Na(+) permeation at positive potentials. However, the N-shaped I-V curve becomes fully developed before the onset of significant slow inactivation, indicating that this N-shaped I-V does not arise from permeability changes associated with entry into slow inactivated states. Thus, changes in the relative contributions of K(+) and Na(+) ions to outward currents could arise either: (a) from depletions of [K(+)](i) sufficient to permit increased Na(+) permeation, or (b) from voltage-dependent changes in K(+) and Na(+) permeabilities. Our results rule out the first of these mechanisms. Furthermore, effects of changing [K(+)](i) and [K(+)](o) on ramp I-V waveforms suggest that applied potential directly affects relative permeation by K(+) and Na(+) ions. Therefore, we conclude that the voltage sensitivity of slow inactivation rates arises indirectly as a result of voltage-dependent changes in the ion occupancy of these channels, and demonstrate that simple barrier models can predict such voltage-dependent changes in relative permeabilities.     

NH2-terminal inactivation peptide binding to C-type-inactivated Kv channels

In many voltage-gated K(+) channels, N-type inactivation significantly accelerates the onset of C-type inactivation, but effects on recovery from inactivation are small or absent. We have exploited the Na(+) permeability of C-type-inactivated K(+) channels to characterize a strong interaction between the inactivation peptide of Kv1.4 and the C-type-inactivated state of Kv1.4 and Kv1.5. The presence of the Kv1.4 inactivation peptide results in a slower decay of the Na(+) tail currents normally observed through C-type-inactivated channels, an effective blockade of the peak Na(+) tail current, and also a delay of the peak tail current. These effects are mimicked by addition of quaternary ammonium ions to the pipette-filling solution. These observations support a common mechanism of action of the inactivation peptide and intracellular quaternary ammonium ions, and also demonstrate that the Kv channel inner vestibule is cytosolically exposed before and after the onset of C-type inactivation. We have also examined the process of N-type inactivation under conditions where C-type inactivation is removed, to compare the interaction of the inactivation peptide with open and C-type-inactivated channels. In C-type-deficient forms of Kv1.4 or Kv1.5 channels, the Kv1.4 inactivation ball behaves like an open channel blocker, and the resultant slowing of deactivation tail currents is considerably weaker than observed in C-type-inactivated channels. We present a kinetic model that duplicates the effects of the inactivation peptide on the slow Na(+) tail of C-type-inactivated channels. Stable binding between the inactivation peptide and the C-type-inactivated state results in slower current decay, and a reduction of the Na(+) tail current magnitude, due to slower transition of channels through the Na(+)-permeable states traversed during recovery from inactivation.     

Extracellular sodium interacts with the HERG channel at an outer pore site

Most voltage-gated K(+) currents are relatively insensitive to extracellular Na(+) (Na(+)(o)), but Na(+)(o) potently inhibits outward human ether-a-go-go-related gene (HERG)-encoded K(+) channel current (Numaguchi, H., J.P. Johnson, Jr., C.I. Petersen, and J.R. Balser. 2000. Nat. Neurosci. 3:429-30). We studied wild-type (WT) and mutant HERG currents and used two strategic probes, intracellular Na(+) (Na(+)(i)) and extracellular Ba(2+) (Ba(2+)(o)), to define a site where Na(+)(o) interacts with HERG. Currents were recorded from transfected Chinese hamster ovary (CHO-K1) cells using the whole-cell voltage clamp technique. Inhibition of WT HERG by Na(+)(o) was not strongly dependent on the voltage during activating pulses. Three point mutants in the P-loop region (S624A, S624T, S631A) with intact K(+) selectivity and impaired inactivation each had reduced sensitivity to inhibition by Na(+)(o). Quantitatively similar effects of Na(+)(i) to inhibit HERG current were seen in the WT and S624A channels. As S624A has impaired Na(+)(o) sensitivity, this result suggested that Na(+)(o) and Na(+)(i) act at different sites. Extracellular Ba(2+) (Ba(2+)(o)) blocks K(+) channel pores, and thereby serves as a useful probe of K(+) channel structure. HERG channel inactivation promotes relief of Ba(2+) block (Weerapura, M., S. Nattel, M. Courtemanche, D. Doern, N. Ethier, and T. Hebert. 2000. J. Physiol. 526:265-278). We used this feature of HERG inactivation to distinguish between simple allosteric and pore-occluding models of Na(+)(o) action. A remote allosteric model predicts that Na(+)(o) will speed relief of Ba(2+)(o) block by promoting inactivation. Instead, Na(+)(o) slowed Ba(2+) egress and Ba(2+) relieved Na(+)(o) inhibition, consistent with Na(+)(o) binding to an outer pore site. The apparent affinities of the outer pore for Na(+)(o) and K(+)(o) as measured by slowing of Ba(2+) egress were compatible with competition between the two ions for the channel pore in their physiological concentration ranges. We also examined the role of the HERG closed state in Na(+)(o) inhibition. Na(+)(o) inhibition was inversely related to pulsing frequency in the WT channel, but not in the pore mutant S624A.     

Regulation of Kir channels by intracellular pH and extracellular K(+): mechanisms of coupling

ROMK channels are regulated by internal pH (pH(i)) and extracellular K(+) (K(+)(o)). The mechanisms underlying this regulation were studied in these channels after expression in Xenopus oocytes. Replacement of the COOH-terminal portion of ROMK2 (Kir1.1b) with the corresponding region of the pH-insensitive channel IRK1 (Kir 2.1) produced a chimeric channel (termed C13) with enhanced sensitivity to inhibition by intracellular H(+), increasing the apparent pKa for inhibition by approximately 0.9 pH units. Three amino acid substitutions at the COOH-terminal end of the second transmembrane helix (I159V, L160M, and I163M) accounted for these effects. These substitutions also made the channels more sensitive to reduction in K(+)(o), consistent with coupling between the responses to pH(i) and K(+)(o). The ion selectivity sequence of the activation of the channel by cations was K(+) congruent with Rb(+) > NH(4)(+) > Na(+), similar to that for ion permeability, suggesting an interaction with the selectivity filter. We tested a model of coupling in which a pH-sensitive gate can close the pore from the inside, preventing access of K(+) from the cytoplasm and increasing sensitivity of the selectivity filter to removal of K(+)(o). We mimicked closure of this gate using positive membrane potentials to elicit block by intracellular cations. With K(+)(o) between 10 and 110 mM, this resulted in a slow, reversible decrease in conductance. However, additional channel constructs, in which inward rectification was maintained but the pH sensor was abolished, failed to respond to voltage under the same conditions. This indicates that blocking access of intracellular K(+) to the selectivity filter cannot account for coupling. The C13 chimera was 10 times more sensitive to extracellular Ba(2+) block than was ROMK2, indicating that changes in the COOH terminus affect ion binding to the outer part of the pore. This effect correlated with the sensitivity to inactivation by H(+). We conclude that decreasing pH(I) increases the sensitivity of ROMK2 channels to K(+)(o) by altering the properties of the selectivity filter.     

Block of inactivation-deficient cardiac Na(+) channels by acetyl-KIFMK-amide

The Na(+) channel alpha-subunit contains an IFM motif that is critical for the fast inactivation process. In this study, we sought to determine whether an IFM-containing peptide, acetyl-KIFMK-amide, blocks open cardiac Na(+) channels via the inner cavity. Intracellular acetyl-KIFMK-amide at 2mM elicited a rapid time-dependent block (tau=0.24 ms) of inactivation-deficient human heart Na(+) channels (hNav1.5-L409C/A410W) at +50 mV. In addition, a peptide-induced tail current appeared conspicuously upon repolarization, suggesting that the activation gate cannot close until acetyl-KIFMK-amide is cleared from the open pore. Repetitive pulses (+50 mV for 20 ms at 1Hz) produced a substantial use-dependent block of both peak and tail currents by approximately 65%. A F1760K mutation (hNav1.5-L409C/A410W/F1760K) abolished the use-dependent block by acetyl-KIFMK-amide and hindered the time-dependent block. Competition experiments showed that acetyl-KIFMK-amide antagonized bupivacaine binding. These results are consistent with a model that two acetyl-KIFMK-amide receptors exist in proximity within the Na(+) channel inner cavity.     

Evidence for multiple open and inactivated states of the hKv1.5 delayed rectifier.

The kinetic properties of hKv1.5, a Shaker-related cardiac delayed rectifier, expressed in Ltk- cells were studied. hKv1.5 currents elicited by membrane depolarizations exhibited a delay followed by biphasic activation. The biphasic activation remained after 5-s prepulses to membrane potentials between -80 and -30 mV; however, the relative amplitude of the slow component increased as the prepulse potential approached the threshold of channel activation, suggesting that the second component did not reflect activation from a hesitant state. The decay of tail currents at potentials between -80 and -30 mV was adequately described with a biexponential. The time course of deactivation slowed as the duration of the depolarizing pulse increased. This was due to a relative increase in the slowly decaying component, despite similar initial amplitudes reflecting a similar open probability after 50- and 500-ms prepulses. To further investigate transitions after the initial activated state, we examined the temperature dependence of inactivation. The time constants of slow inactivation displayed little temperature and voltage dependence, but the degree of the inactivation increased substantially with increased temperature. Recovery from inactivation proceeded with a biexponential time course, but long prepulses at depolarized potentials slowed the apparent rate of recovery from inactivation. These data strongly indicate that hKv1.5 has both multiple open states and multiple inactivated states.     

Regulation of a mammalian Shaker-related potassium channel, hKv1.5, by extracellular potassium and pH

Using the whole-cell recording mode of the patch-clamp technique we studied the effects of removal of extracellular potassium, [K(+)](o), on a mammalian Shaker-related K(+) channel, hKv1.5. In the absence of [K(+)](o), current through hKv1.5 was similar to currents obtained in the presence of 4.5 mM [K(+)](o). This observation was not expected as earlier results had suggested that either positively charged residues or the presence of a nitrogen-containing residue at the external TEA(+) binding site (R487 in hKv1.5) caused current loss upon removal of [K(+)](o). However, the current loss in hKv1.5 was observed when the extracellular pH, pH(o), was reduced from 7.4 to 6.0, a behavior similar to that observed previously for current through mKv1.3 with a histidine at the equivalent position (H404). These observations suggested that the charge at R487 in hKv1.5 channels was influenced by other amino acids in the vicinity. Replacement of a histidine at position 463 in hKv1.5 by glycine confirmed this hypothesis making this H463G mutant channel sensitive to removal of [K(+)](o) even at pH(o) 7.4. We conclude that the protonation of H463 at pH 7.4 might induce a pK(a) shift of R487 that influences the effective charge at this position leading to a not fully protonated arginine. Furthermore, we assume that the charge at position 487 in hKv1.5 can directly or indirectly disturb the occupation of a K(+) binding site within the channel pore possibly by electrostatic interaction. This in turn might interfere with the concerted transition of K(+) ions resulting in a loss of K(+) conduction.     

Protons block BK channels by competitive inhibition with K+ and contribute to the limits of unitary currents at high voltages

Proton block of unitary currents through BK channels was investigated with single-channel recording. Increasing intracellular proton concentration decreased unitary current amplitudes with an apparent pKa of 5.1 without discrete blocking events, indicating fast proton block. Unitary currents recorded at pH(i) 8.0 and 9.0 had the same amplitudes, indicating that 10(-8) M H(+) had little blocking effect. Increasing H(+) by recording at pH(i) 7.0, 6.0, and 5.0 then reduced the unitary currents by 13%, 25%, and 53%, respectively, at +200 mV. Increasing K(+)(i) relieved the proton block in a manner consistent with competitive inhibition of K(+)(i) action by H(+)(i). Proton block was voltage dependent, increasing with depolarization, indicating that block was coupled to the electric field of the membrane. Proton block was not described by the Woodhull equation for noncompetitive voltage-dependent block, but was described by an equation for cooperative competitive inhibition that included voltage-dependent block from the Woodhull equation. Proton block was still present after replacing the eight negative charges in the ring of charge at the entrance to the intracellular vestibule by uncharged amino acids. Thus, the ring of charge is not the site of proton block or of competitive inhibition of K(+)(i) action by H(+)(i). With 150 mM symmetrical KCl, unitary current amplitudes increased with depolarization, reaching 66 pA at +350 mV (pH(i) 7.0). The increase in amplitude with voltage became sublinear for voltages >100 mV. The sublinearity was unaffected by removing from the intracellular solutions Ca(2+) and Ba(2+) ions, the Ca(2+) buffers EGTA and HEDTA, the pH buffer TES, or by replacing Cl(-) with MeSO(3)(-). Proton block accounted for approximately 40% of the sublinearity at +200 mV and pH 7.0, indicating that factors in addition to proton block contribute to the sublinearity of the unitary currents through BK channels.     

Quaternary organic amines inhibit Na,K pump current in a voltage-dependent manner: direct evidence of an extracellular access channel in the Na,K-ATPase

The effects of organic quaternary amines, tetraethylammonium (TEA) chloride and benzyltriethylammonium (BTEA) chloride, on Na,K pump current were examined in rat cardiac myocytes superfused in extracellular Na(+)-free solutions and whole-cell voltage-clamped with patch electrodes containing a high Na(+)-salt solution. Extracellular application of these quaternary amines competitively inhibited extracellular K(+) (K(+)(o)) activation of Na,K pump current; however, the concentration for half maximal inhibition of Na,K pump current at 0 mV (K(0)(Q)) by BTEA, 4.0 +/- 0.3 mM, was much lower than the K(0)(Q) for TEA, 26.6 +/- 0.7 mM. Even so, the fraction of the membrane electric field dissipated during K(+)(o) activation of Na,K pump current (lambda(K)), 39 +/- 1%, was similar to lambda(K) determined in the presence of TEA (37 +/- 2%) and BTEA (35 +/- 2%), an indication that the membrane potential (V(M)) dependence for K(+)(o) activation of the Na,K pump current was unaffected by TEA and BTEA. TEA was found to inhibit the Na,K pump current in a V(M)-independent manner, i.e., inhibition of current dissipated 4 +/- 2% of the membrane electric field. In contrast, BTEA dissipated 40 +/- 5% of the membrane electric field during inhibition of Na,K pump current. Thus, BTEA inhibition of the Na,K-ATPase is V(M)-dependent. The competitive nature of inhibition as well as the similar fractions of the membrane electric field dissipated during K(+)(o)-dependent activation and BTEA-dependent inhibition of Na,K pump current suggest that BTEA inhibits the Na,K-ATPase at or very near the enzyme's K(+)(o) binding site(s) located in the membrane electric field. Given previous findings that organic quaternary amines are not occluded by the Na,K-ATPase, these data clearly demonstrate that an ion channel-like structure provides access to K(+)(o) binding sites in the enzyme.     

Gating charge immobilization caused by the transition between inactivated states in the Kv1.5 channel

Sustained Na(+) or Li(+) conductance is a feature of the inactivated state in wild-type (WT) and nonconducting Shaker and Kv1.5 channels, and has been used here to investigate the cause of off-gating charge immobilization in WT and Kv1.5-W472F nonconducting mutant channels. Off-gating immobilization in response to brief pulses in cells perfused with NMG/NMG is the result of a more negative voltage dependence of charge recovery (V(1/2) is -96 mV) compared with on-gating charge movement (V(1/2) is -6.3 mV). This shift is known to be associated with slow inactivation in Shaker channels and the disparity is reduced by 40 mV, or approximately 50% in the presence of 135 mM Cs. Off-gating charge immobilization is voltage-dependent with a V(1/2) of -12 mV, and correlates well with the development of Na(+) conductance on repolarization through C-type inactivated channels (V(1/2) is -11 mV). As well, the time-dependent development of the inward Na(+) tail current and gating charge immobilization after depolarizing pulses of different durations has the same time constant (tau = 2.7 ms). These results indicate that in Kv1.5 channels the transition to a stable C-type inactivated state takes only 2-3 ms and results in strong charge immobilization in the absence of Group IA metal cations, or even in the presence of Na. Inclusion of low concentrations of Cs delays the appearance of Na(+) tail currents in WT channels, prevents transition to inactivated states in Kv1.5-W472F nonconducting mutant channels, and removes charge immobilization. Higher concentrations of Cs are able to modulate the deactivating transition in Kv1.5 channels and prevent the residual slowing of charge return.     

Direct block of cloned hKv1.5 channel by cytochalasins, actin-disrupting agents

The action of cytochalasins, actin-disrupting agents on human Kv1.5 channel (hKv1.5) stably expressed in Ltk^– cells was investigated using the whole cell patch-clamp technique. Cytochalasin B inhibited hKv1.5 currents rapidly and reversibly at +60 mV in a concentration-dependent manner with an IC₅₀ of 4.2 µM. Cytochalasin A, which has a structure very similar to cytochalasin B, inhibited hKv1.5 (IC₅₀ of 1.4 µM at +60 mV). Pretreatment with other actin filament disruptors cytochalasin D and cytochalasin J, and an actin filament stabilizing agent phalloidin had no effect on the cytochalasin B-induced inhibition of hKv1.5 currents. Cytochalasin B accelerated the decay rate of inactivation for the hKv1.5 currents. Cytochalasin B-induced inhibition of the hKv1.5 channels was voltage dependent with a steep increase over the voltage range of the channel's opening. However, the inhibition exhibited voltage independence over the voltage range in which channels are fully activated. Cytochalasin B produced no significant effect on the steady-state activation or inactivation curves. The rate constants for association and dissociation of cytochalasin B were 3.7 µM/s and 7.5 s^–1, respectively. Cytochalasin B produced a use-dependent inhibition of hKv1.5 current that was consistent with the slow recovery from inactivation in the presence of the drug. Cytochalasin B (10 µM) also inhibited an ultrarapid delayed rectifier K⁺ current (I_K,ur) in human atrial myocytes. These results indicate that cytochalasin B primarily blocks activated hKv1.5 channels and endogenous I_K,ur in a cytoskeleton-independent manner as an open-channel blocker. voltage-gated K⁺ channel; heart; open channel block     

Increased excitability of acidified skeletal muscle: role of chloride conductance

Generation of the action potentials (AP) necessary to activate skeletal muscle fibers requires that inward membrane currents exceed outward currents and thereby depolarize the fibers to the voltage threshold for AP generation. Excitability therefore depends on both excitatory Na+ currents and inhibitory K+ and Cl- currents. During intensive exercise, active muscle loses K+ and extracellular K+ ([K+]o) increases. Since high [K+]o leads to depolarization and ensuing inactivation of voltage-gated Na+ channels and loss of excitability in isolated muscles, exercise-induced loss of K+ is likely to reduce muscle excitability and thereby contribute to muscle fatigue in vivo. Intensive exercise, however, also leads to muscle acidification, which recently was shown to recover excitability in isolated K(+)-depressed muscles of the rat. Here we show that in rat soleus muscles at 11 mM K+, the almost complete recovery of compound action potentials and force with muscle acidification (CO2 changed from 5 to 24%) was associated with reduced chloride conductance (1731 +/- 151 to 938 +/- 64 microS/cm2, P < 0.01) but not with changes in potassium conductance (405 +/- 20 to 455 +/- 30 microS/cm2, P < 0.16). Furthermore, acidification reduced the rheobase current by 26% at 4 mM K+ and increased the number of excitable fibers at elevated [K+]o. At 11 mM K+ and normal pH, a recovery of excitability and force similar to the observations with muscle acidification could be induced by reducing extracellular Cl- or by blocking the major muscle Cl- channel, ClC-1, with 30 microM 9-AC. It is concluded that recovery of excitability in K(+)-depressed muscles induced by muscle acidification is related to reduction in the inhibitory Cl- currents, possibly through inhibition of ClC-1 channels, and acidosis thereby reduces the Na+ current needed to generate and propagate an AP. Thus short term regulation of Cl- channels is important for maintenance of excitability in working muscle.     

External nickel blocks human Kv1.5 channels stably expressed in CHO cells

We have investigated the actions of Nickel (Ni²⁺) on a human cardiac potassium channel (hKv1.5), the main component of human atrial ultra-rapid delayed rectifier current, stably expressed in Chinese hamster ovary cell line using the whole-cell voltage-clamp technique. External Ni²⁺ reversibly decreased the amplitude of the current in a concentration-dependent manner. The concentration for half-maximum inhibition of the current at +50 mV was 568 μm. The activation, deactivation, reactivation kinetics of the current were not affected by Ni²⁺. Block was not voltage-dependent but frequency-dependent block was apparent. The extent of channel block during the first pulse increased when the duration of exposure to Ni²⁺, prior to channel activation, was prolonged indicating that Ni²⁺ interacted with hKv1.5 in the closed state. The percentage of current remaining in presence of Ni²⁺ decreased steeply over the range of steady-state channel inactivation, consistent with an enhanced block with increased inactivation. This suggests that Ni²⁺ preferentially blocks nonconducting hKv1.5 channels, either in the resting or inactivated state in a concentration-dependent manner. The data indicate that the mechanisms of hKv1.5 channel inhibition by Ni²⁺ are distinct from those of other K⁺ channels. Received: 12 October 2000/Revised: 14 May 2001     

Na self inhibition of human epithelial Na channel: temperature dependence and effect of extracellular proteases

The regulation of the open probability of the epithelial Na(+) channel (ENaC) by the extracellular concentration of Na(+), a phenomenon called "Na(+) self inhibition," has been well described in several natural tight epithelia, but its molecular mechanism is not known. We have studied the kinetics of Na(+) self inhibition on human ENaC expressed in Xenopus oocytes. Rapid removal of amiloride or rapid increase in the extracellular Na(+) concentration from 1 to 100 mM resulted in a peak inward current followed by a decline to a lower quasi-steady-state current. The rate of current decline and the steady-state level were temperature dependent and the current transient could be well explained by a two-state (active-inactive) model with a weakly temperature-dependent (Q(10)act = 1.5) activation rate and a strongly temperature-dependant (Q(10)inact = 8.0) inactivation rate. The steep temperature dependence of the inactivation rate resulted in the paradoxical decrease in the steady-state amiloride-sensitive current at high temperature. Na(+) self inhibition depended only on the extracellular Na(+) concentration but not on the amplitude of the inward current, and it was observed as a decrease of the conductance at the reversal potential for Na(+) as well as a reduction of Na(+) outward current. Self inhibition could be prevented by exposure to extracellular protease, a treatment known to activate ENaC or by treatment with p-CMB. After protease treatment, the amiloride-sensitive current displayed the expected increase with rising temperature. These results indicate that Na(+) self inhibition is an intrinsic property of sodium channels resulting from the expression of the alpha, beta, and gamma subunits of human ENaC in Xenopus oocyte. The extracellular Na(+)-dependent inactivation has a large energy of activation and can be abolished by treatment with extracellular proteases.     

Hydrogen sulfide is a partially redox-independent activator of the human jejunum Na+ channel, Nav1.5

Hydrogen sulfide (H(2)S) is produced endogenously by L-cysteine metabolism. H(2)S modulates several ion channels with an unclear mechanism of action. A possible mechanism is through reduction-oxidation reactions attributable to the redox potential of the sulfur moiety. The aims of this study were to determine the effects of the H(2)S donor NaHS on Na(V)1.5, a voltage-dependent sodium channel expressed in the gastrointestinal tract in human jejunum smooth muscle cells and interstitial cells of Cajal, and to elucidate whether H(2)S acts on Na(V)1.5 by redox reactions. Whole cell Na(+) currents were recorded in freshly dissociated human jejunum circular myocytes and Na(V)1.5-transfected human embryonic kidney-293 cells. RT-PCR amplified mRNA for H(2)S enzymes cystathionine β-synthase and cystathionine γ-lyase from the human jejunum. NaHS increased native Na(+) peak currents and shifted the half-point (V(1/2)) of steady-state activation and inactivation by +21 ± 2 mV and +15 ± 3 mV, respectively. Similar effects were seen on the heterologously expressed Na(V)1.5 α subunit with EC(50)s in the 10(-4) to 10(-3) M range. The reducing agent dithiothreitol (DTT) mimicked in part the effects of NaHS by increasing peak current and positively shifting steady-state activation. DTT together with NaHS had an additive effect on steady-state activation but not on peak current, suggesting that the latter may be altered via reduction. Pretreatment with the Hg(2+)-conjugated oxidizer thimerosal or the alkylating agent N-ethylmaleimide inhibited or decreased NaHS induction of Na(V)1.5 peak current. These studies show that H(2)S activates the gastrointestinal Na(+) channel, and the mechanism of action of H(2)S is partially redox independent.     

Time-dependent block and resurgent tail currents induced by mouse beta4(154-167) peptide in cardiac Na+ channels

Resurgent tail Na(+) currents were first discovered in cerebellar Purkinje neurons. A recent study showed that a 14-mer fragment of a mouse beta4 subunit, beta4(154-167), acts as an intracellular open-channel blocker and elicits resurgent currents in Purkinje neurons (Grieco, T.M., J.D. Malhotra, C. Chen, L.L. Isom, and I.M. Raman. 2005. Neuron. 45:233-244). To explore these phenotypes in vitro, we characterized beta4(154-167) actions in inactivation-deficient cardiac hNav1.5 Na(+) channels expressed in human embryonic kidney 293t cells. Intracellular beta4(154-167) from 25-250 microM elicited a conspicuous time-dependent block of inactivation-deficient Na(+) currents at 50 mV in a concentration-dependent manner. On and off rates for beta4(154-167) binding were estimated at 10.1 microM(-1)s(-1) and 49.1 s(-1), respectively. Upon repolarization, large tail currents emerged with a slight delay at -140 mV, probably as a result of the rapid unblocking of beta4(154-167). Near the activation threshold (approximately -70 mV), resurgent tail currents were robust and long lasting. Likewise, beta4(154-167) induces resurgent currents in wild-type hNav1.5 Na(+) channels, although to a lesser extent. The inactivation peptide acetyl-KIFMK-amide not only restored the fast inactivation phenotype in hNav1.5 inactivation-deficient Na(+) channels but also elicited robust resurgent currents. When modified by batrachotoxin (BTX), wild-type hNav1.5 Na(+) channels opened persistently but became resistant to beta4(154-167) and acetyl-KIFMK-amide block. Finally, a lysine substitution of a phenylalanine residue at D4S6, F1760, which forms a part of receptors for local anesthetics and BTX, rendered cardiac Na(+) channels resistant to beta4(154-167). Together, our in vitro studies identify a putative S6-binding site for beta4(154-167) within the inner cavity of hNav1.5 Na(+) channels. Such an S6 receptor readily explains (1) why beta4(154-167) gains access to its receptor as an open-channel blocker, (2), why bound beta4(154-167) briefly prevents the activation gate from closing by a "foot-in-the-door" mechanism during deactivation, (3) why BTX inhibits beta4(154-167) binding by physical exclusion, and (4) why a lysine substitution of residue F1760 eliminates beta4(154-167) binding.