A Model of the Block of Voltage-Gated Potassium Kv4.2 Ionic Currents by 4-Aminopyridine.

Voltage clamp recordings of macroscopic currents were made from rat potassium-gated potassium 4.2(Kv4.2) channels expressed in human embryonic kidney (HEK293) cells with the main goals of quantifying the concentration, time, and voltage dependence of the block and to generate a state model that replicates the features of the block. When applied either externally or internally, the block of Kv4.2 currents by 4-aminopyridine (4AP) occurs at the holding potential (-80 mV), is affected by the stimulus frequency, and is relieved by membrane depolarization. The Kd for the tonic block at -80 mV was 0.9 ± 0.07 mM and was consistent with 1:1 binding. Relief of block during a step to 50 mV was well fitted by a single exponential with a time constant of ∼40 milliseconds. At -80 mV, the association rate constant was 0.08 mM-1 s-1, and the off-rate was 0.08 s-1 The state model replicates the features of the experimental data reasonably well by assuming that 4AP binds only to closed states, that 4AP binding and inactivation are mutually exclusive processes, and that the activation of closed-bound channels is the same as for closed channels. Since the open channel has a very low or no affinity for 4AP, channel opening promotes the unbinding of 4AP, which accounts for the reverse use dependence of the block.

ShakerIR and Kv1.5 mutant channels with enhanced slow inactivation also exhibit K⁺ o-dependent resting inactivation.

Previous studies have shown that in N-type inactivation-removed Shaker (ShakerIR) channels, the T449K and T449A mutations result in enhanced slow inactivation. These mutant channels also show a loss of conductance in 0 mM K⁺ o that was attributed to an inactivation process occurring from the closed, resting state and which we refer to as resting inactivation. Similar behavior has also been observed in the Kv1.5 H463G mutant channel. To date, the time courses for the onset of and recovery from resting inactivation have been unknown, but a comparison of the kinetics for resting inactivation induced at -80 mV and slow inactivation evoked at +50 mV may provide information on whether these two processes are mechanistically related. Here, we present an analysis of the time courses for the onset of and recovery from [K⁺]o-dependent resting inactivation and depolarization-induced inactivation of these mutant channels. Despite the enhancement of slow inactivation in the ShakerIR T449K, T449A, and Kv1.5 H463G mutants, the time constant for slow inactivation at +50 mV (τ inact) was relatively insensitive to the increases or decreases of [K(+)]o, confirming that accelerated inactivation from the open state does not underlie the loss of conductance in 0 mM K⁺. For all three mutants, the time constant for resting inactivation (τ RI), induced by exposure to 0 mM K⁺ o solution at -80 mV, was at least an order of magnitude larger than τ inact. On the other hand, the time course of recovery at -80 mV of each mutant from 0 mM K(+) o-induced resting inactivation was the same as that from depolarization-induced slow inactivation. This latter result suggests that the 0 mM K⁺ o-induced resting inactivation of these mutant ShakerIR and Kv1.5 channels is mechanistically related to slow inactivation.

External Ba(2+) block of Kv4.2 channels is enhanced in the closed-inactivated state.

The effect of external barium ions on rat Kv4.2 channels expressed in HEK293 cells was investigated using whole cell, voltage-clamp recordings to determine its mechanism of action as well as its usefulness as a tool to probe the permeation pathway. Ba(2+) caused a concentration-dependent inhibition of current that was antagonized by increasing the external concentration of K(+) ([K(+)](o)), and the concentration and time dependence of the inhibition were well fitted by a model involving two binding sites aligned in series. Recovery from current inhibition was enhanced by increasing the intensity, duration, or frequency of depolarizing steps or by increasing [K(+)](o). These properties are consistent with the conclusion that Ba(2+) is a permeant ion that, by virtue of a stable interaction with a deep pore site, is able to block conduction. This blocking action was subsequently exploited to gain insights into the pore configuration in different channel states. In addition to blocking one or more states populated by brief depolarizing pulses to 80 mV, Ba(2+) blocked closed channels [the membrane voltage (V(m)) = -80 mV] and closed-inactivated channels (V(m) = -40 mV). Interestingly, the block of closed-inactivated channels was faster and more complete than for closed channels, which we interpret to mean that conformational changes underlying closed-state inactivation (CSI) enhance Ba(2+) binding and that the outer pore mouth remains patent during CSI. This provides the first direct evidence that an inactivation process involving a constriction of the outer pore mouth does not account for CSI in Kv4.2.

Kinetic analysis of the effects of H+ or Ni2+ on Kv1.5 current shows that both ions enhance slow inactivation and induce resting inactivation.

External H+ and Ni2+ ions inhibit Kv1.5 channels by increasing current decay during a depolarizing pulse and reducing the maximal conductance. Although the former may be attributed to an enhancement of slow inactivation occurring from the open state, the latter cannot. Instead, we propose that the loss of conductance is due to the induction, by H+ or Ni2+, of a resting inactivation process. To assess whether the two inactivation processes are mechanistically related, we examined the time courses for the onset of and recovery from H+- or Ni2+-enhanced slow inactivation and resting inactivation. Compared to the time course of H+- or Ni2+-enhanced slow inactivation at +50 mV, the onset of resting inactivation induced at 80 mV with either ion involves a relatively slower process. Recovery from slow inactivation under control conditions was bi-exponential, indicative of at least two inactivated states. Recovery following H+- or Ni2+-enhanced slow inactivation or resting inactivation had time constants similar to those for recovery from control slow inactivation, although H+ and Ni2+ biased inactivation towards states from which recovery was fast and slow, respectively. The shared time constants suggest that the H+- and Ni2+-enhanced slow inactivated and induced resting inactivated states are similar to those visited during control slow inactivation at pH 7.4. We conclude that in Kv1.5 H+ and Ni2+ differentially enhance a slow inactivation process that involves at least two inactivated states and that resting inactivation is probably a close variant of slow inactivation.

Control of voltage-gated K+ channel permeability to NMDG+ by a residue at the outer pore.

Crystal structures of potassium (K(+)) channels reveal that the selectivity filter, the narrow portion of the pore, is only approximately 3-A wide and buttressed from behind, so that its ability to expand is highly constrained, and the permeation of molecules larger than Rb(+) (2.96 A in diameter) is prevented. N-methyl-d-glucamine (NMDG(+)), an organic monovalent cation, is thought to be a blocker of Kv channels, as it is much larger (approximately 7.3 A in mean diameter) than K(+) (2.66 A in diameter). However, in the absence of K(+), significant NMDG(+) currents could be recorded from human embryonic kidney cells expressing Kv3.1 or Kv3.2b channels and Kv1.5 R487Y/V, but not wild-type channels. Inward currents were much larger than outward currents due to the presence of intracellular Mg(2+) (1 mM), which blocked the outward NMDG(+) current, resulting in a strong inward rectification. The NMDG(+) current was inhibited by extracellular 4-aminopyridine (5 mM) or tetraethylammonium (10 mM), and largely eliminated in Kv3.2b by an S6 mutation that prevents the channel from opening (P468W) and by a pore helix mutation in Kv1.5 R487Y (W472F) that inactivates the channel at rest. These data indicate that NMDG(+) passes through the open ion-conducting pore and suggest a very flexible nature of the selectivity filter itself. 0.3 or 1 mM K(+) added to the external NMDG(+) solution positively shifted the reversal potential by approximately 16 or 31 mV, respectively, giving a permeability ratio for K(+) over NMDG(+) (P(K)(+)/P(NMDG)(+)) of approximately 240. Reversal potential shifts in mixtures of K(+) and NMDG(+) are in accordance with P(K)(+)/P(NMDG)(+), indicating that the ions compete for permeation and suggesting that NMDG(+) passes through the open state. Comparison of the outer pore regions of Kv3 and Kv1.5 channels identified an Arg residue in Kv1.5 that is replaced by a Tyr in Kv3 channels. Substituting R with Y or V allowed Kv1.5 channels to conduct NMDG(+), suggesting a regulation by this outer pore residue of Kv channel flexibility and, as a result, permeability.

Closed-state inactivation induced in K(V)1 channels by extracellular acidification.

Extracellular acidification regulates the biophysical properties of many voltage-gated potassium channels. Most often acidic pH reduces peak current and enhances current decay during depolarization. Here we review recent data from single channel and voltage clamp fluorimetry studies, which suggest that these two effects of protons are mediated by distinct kinetic processes. This new mechanistic insight directly demonstrates that whilst the enhanced decay of current observed with acidic pH is due to an accelerated entry of open channels into P/C-type inactivation, the main mechanism for the reduction in peak channel conductance is a stabilization of resting channels in closed-inactivated states. Thus acidic pH acts to reduce the mean burst time of conducting channels, as well as to prevent other channels from opening at all, and in so doing, reveals that both open- and closed-state inactivation processes can co-exist in K(V) channels.

External Ba2+ block of human Kv1.5 at neutral and acidic pH: evidence for Ho+-induced constriction of the outer pore mouth at rest.

Previous studies have shown that low pHo accelerates depolarization-induced inactivation and decreases the macroscopic conductance by reducing channel availability. To test the hypothesis that outer pore constriction underlies the decreased conductance at low pHo, external Ba2+ was used to examine the accessibility of the channel pore at rest under neutral and acidic conditions. At pHo 7.4, Ba2+ block of closed channels follows a monoexponential time course and involves a low-affinity superficial site (KD congruent with 1 mM, -80 mV, 0 mM Ko(+)) and a high-affinity site (KD congruent with 4 microM) deeper in the pore. Depolarization promotes Ba2+ dissociation and an analytical model incorporating state-dependent changes of Ba2+ affinity is presented that replicates the frequency dependence of the time course and the extent of block. Open-channel block by Ba2+ is weak. At pHo 5.5, both the access to and exit from the deep site is inhibited. These results are consistent with a low-pHo-induced conformational change in the outer pore that prevents Ba2+ binding at rest or unbinding during depolarization. If a pore constriction is involved, similar to that proposed to occur during P/C-type inactivation, this would imply that closed-state inactivation in Kv1.5 occurs under acidic conditions.

An activation gating switch in Kv1.2 is localized to a threonine residue in the S2-S3 linker.

The activation properties of Kv1.2 channels are highly variable, with reported half-activation (V((1/2))) values ranging from approximately -40 mV to approximately +30 mV. Here we show that this arises because Kv1.2 channels occupy two distinct gating modes ("fast" and "slow"). "Slow" gating (tau(act) = 90 +/- 6 ms at +35 mV) was associated with a V((1/2)) of activation of +16.6 +/- 1.1 mV, whereas "fast" gating (tau(act) = 4.5 +/- 1.7 ms at +35 mV) was associated with a V((1/2)) of activation of -18.8 +/- 2.3 mV. It was possible to switch between gating modes by applying a prepulse, which suggested that channels activate to a single open state along separate "fast" and "slow" activation pathways. Using chimeras and point mutants between Kv1.2 and Kv1.5 channels, we determined that introduction of a positive charge at or around threonine 252 in the S2-S3 linker of Kv1.2 abolished "slow" activation gating. Furthermore, dialysis of the cytoplasm or excision of cell-attached patches from cells expressing Kv1.2 channels switched gating from "slow" to "fast", suggesting involvement of cytoplasmic regulators. Collectively, these results demonstrate two modes of activation gating in Kv1.2 and specific residues in the S2-S3 linker that act as a switch between these modes.

A direct demonstration of closed-state inactivation of K+ channels at low pH.

Lowering external pH reduces peak current and enhances current decay in Kv and Shaker-IR channels. Using voltage-clamp fluorimetry we directly determined the fate of Shaker-IR channels at low pH by measuring fluorescence emission from tetramethylrhodamine-5-maleimide attached to substituted cysteine residues in the voltage sensor domain (M356C to R362C) or S5-P linker (S424C). One aspect of the distal S3-S4 linker alpha-helix (A359C and R362C) reported a pH-induced acceleration of the slow phase of fluorescence quenching that represents P/C-type inactivation, but neither site reported a change in the total charge movement at low pH. Shaker S424C fluorescence demonstrated slow unquenching that also reflects channel inactivation and this too was accelerated at low pH. In addition, however, acidic pH caused a reversible loss of the fluorescence signal (pKa = 5.1) that paralleled the reduction of peak current amplitude (pKa = 5.2). Protons decreased single channel open probability, suggesting that the loss of fluorescence at low pH reflects a decreased channel availability that is responsible for the reduced macroscopic conductance. Inhibition of inactivation in Shaker S424C (by raising external K(+) or the mutation T449V) prevented fluorescence loss at low pH, and the fluorescence report from closed Shaker ILT S424C channels implied that protons stabilized a W434F-like inactivated state. Furthermore, acidic pH changed the fluorescence amplitude (pKa = 5.9) in channels held continuously at -80 mV. This suggests that low pH stabilizes closed-inactivated states. Thus, fluorescence experiments suggest the major mechanism of pH-induced peak current reduction is inactivation of channels from closed states from which they can activate, but not open; this occurs in addition to acceleration of P/C-type inactivation from the open state.

SCAM analysis reveals a discrete region of the pore turret that modulates slow inactivation in Kv1.5.

In Kv1.5, protonation of histidine 463 in the S5-P linker (turret) increases the rate of depolarization-induced inactivation and decreases the peak current amplitude. In this study, we examined how amino acid substitutions that altered the physico-chemical properties of the side chain at position 463 affected slow inactivation and then used the substituted cysteine accessibility method (SCAM) to probe the turret region (E456-P468) to determine whether residue 463 was unique in its ability to modulate the macroscopic current. Substitutions at position 463 of small, neutral (H463G and H463A) or large, charged (H463R, H463K, and H463E) side groups accelerated inactivation and induced a dependency of the current amplitude on the external potassium concentration. When cysteine substitutions were made in the distal turret (T462C-P468C), modification with either the positively charged [2-(trimethylammonium)ethyl] methanethiosulfonate bromide (MTSET) or negatively charged sodium (2-sulfonatoethyl) methanethiosulfonate reagent irreversibly inhibited current. This inhibition could be antagonized either by the R487V mutation (homologous to T449V in Shaker) or by raising the external potassium concentration, suggesting that current inhibition by MTS reagents resulted from an enhancement of inactivation. These results imply that protonation of residue 463 does not modulate inactivation solely by an electrostatic interaction with residues near the pore mouth, as proposed by others, and that residue 463 is part of a group of residues within the Kv1.5 turret that can modulate P/C-type inactivation.

4-aminopyridine prevents the conformational changes associated with p/c-type inactivation in shaker channels.

The effect of 4-aminopyridine (4-AP) on Kv channel activation has been extensively investigated, but its interaction with inactivation is less well understood. Voltage-clamp fluorimetry was used to directly monitor the action of 4-AP on conformational changes associated with slow inactivation of Shaker channels. Tetramethylrhodamine-5-maleimide was used to fluorescently label substituted cysteine residues in the S3-S4 linker (A359C) and pore (S424C). Activation- and inactivation-induced changes in fluorophore microenvironment produced fast and slow phases of fluorescence that were modified by 4-AP. In Shaker A359C, 4-AP block reduced the slow-phase contribution from 61 +/- 3 to 28 +/- 5%, suggesting that binding inhibits the conformational changes associated with slow inactivation and increased the fast phase that reports channel activation from 39 +/- 3 to 72 +/- 5%. In addition, 4-AP enhanced both fast and slow phases of fluorescence return upon repolarization (tau reduced from 87 +/- 15 to 40 +/- 1 ms and from 739 +/- 83 to 291 +/- 21 ms, respectively), suggesting that deactivation and recovery from inactivation were enhanced. In addition, the effect of 4-AP on the slow phase of fluorescence was dramatically reduced in channels with either reduced (T449V) or permanent P-type (W434F) inactivation. Interestingly, the slow phase of fluorescence return of W434F channels was enhanced by 4-AP, suggesting that 4-AP prevents the transition to C-type inactivation in these channels. These data directly demonstrate that 4-AP prevents slow inactivation of Kv channels and that 4-AP can bind to P-type-inactivated channels and selectively inhibit the onset of C-type inactivation.

Block by internal Mg2+ causes voltage-dependent inactivation of Kv1.5.

Internal Mg2+ blocks many potassium channels including Kv1.5. Here, we show that internal Mg2+ block of Kv1.5 induces voltage-dependent current decay at strongly depolarised potentials that contains a component due to acceleration of C-type inactivation after pore block. The voltage-dependent current decay was fitted to a bi-exponential function (tau(fast) and tau(slow)). Without Mg2+, tau(fast) and tau(slow) were voltage-independent, but with 10 mM Mg2+, tau(fast) decreased from 156 ms at +40 mV to 5 ms at +140 mV and tau(slow) decreased from 2.3 s to 206 ms. With Mg2+, tail currents after short pulses that allowed only the fast phase of decay showed a rising phase that reflected voltage-dependent unbinding. This suggested that the fast phase of voltage-dependent current decay was due to Mg2+ pore block. In contrast, tail currents after longer pulses that allowed the slow phase of decay were reduced to almost zero suggesting that the slow phase was due to channel inactivation. Consistent with this, the mutation R487V (equivalent to T449V in Shaker) or increasing external K+, both of which reduce C-type inactivation, prevented the slow phase of decay. These results are consistent with voltage-dependent open-channel block of Kv1.5 by internal Mg2+ that subsequently induces C-type inactivation by restricting K+ filling of the selectivity filter from the internal solution.

Single channel analysis reveals different modes of Kv1.5 gating behavior regulated by changes of external pH.

In the voltage-gated potassium channel Kv1.5, extracellular acidification decreases the peak macroscopic conductance and accelerates slow inactivation. To better understand the mechanistic basis for these two effects, we recorded unitary currents of Kv1.5 expressed in a mouse cell line (ltk-) using the voltage clamp technique both in cell-attached and excised outside-out patches. Single channel current amplitude at 100 mV (1.7 +/- 0.2 pA at pH 7.4, 1.7 +/- 0.2 pA at pH 6.4) and the single channel conductance between 0 and 100 mV (11.8 +/- 0.6 pS at pH 7.4 and 11.3 +/- 0.8 pS at pH 6.4) did not change significantly with pH. External acidification significantly decreased the number of active sweeps, and this reduction in channel availability accounted for most of the reduction of the peak macroscopic current. The results of runs analyses suggested the null sweeps occur in clusters, and the rate constants for the transition between clusters of null and active sweeps at pH 6.4 were slow (0.12 and 0.18 s(-1), to and from the active clusters, respectively). We propose that low pH facilitates a shift from an available mode (mode A) into an unavailable mode of gating (mode U). In addition to promoting mode U gating, external acidification accelerates depolarization-induced inactivation, which is manifest at the single channel level as a reduction of the mean burst length and an apparent increase of the interburst interval. These effects of external acidification, which are thought to reflect the protonation of a histidine residue in the turret (H-463), point to an important role for the turret in the regulation of channel availability and inactivation.

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 appearance 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 mechanism (i.e., by increasing channel inactivation, which occurs in the resting state or develops very rapidly after activation).

Synergistic inhibition of the maximum conductance of Kv1.5 channels by extracellular K+ reduction and acidification.

Voltage-gated potassium (Kv) channels exist in the membranes of all living cells. Of the functional classes of Kv channels, the Kv1 channels are the largest and the best studied and are known to play essential roles in excitable cell function, providing an essential counterpoint to the various inward currents that trigger excitability. The serum potassium concentration [K(+)(o)] is tightly regulated in mammals and disturbances can cause significant functional alterations in the electrical behavior of excitable tissues in the nervous system and the heart. At least some of these changes may be mediated by Kv channels that are regulated by changes in the extracellular K(+) concentration. As well as changes in serum [K(+)(o)], tissue acidification is a frequent pathological condition known to inhibit Shaker and Kv1 voltage-gated potassium channels. In recent studies, it has become recognized that the acidification-induced inhibition of some Kv1 channels is K(+)(o)-dependent, and the suggestion has been made that pH and K(+)(o) may regulate the channels via a common mechanism. Here we discuss P/C type inactivation as the common pathway by which some Kv channels become unavailable at acid pH and lowered K(+)(o). It is suggested that binding of protons to a regulatory site in the outer pore mouth of some Kv channels favors transitions to the inactivated state, whereas K(+) ions exert countereffects. We suggest that modulation of the number of excitable voltage-gated K(+) channels in the open vs inactivated states of the channels by physiological H(+) and K(+) concentrations represents an important pathway to control Kv channel function in health and disease.

The external K+ concentration and mutations in the outer pore mouth affect the inhibition of kv1.5 current by Ni2+.

By examining the consequences both of changes of [K+]o and of point mutations in the outer pore mouth, our goal was to determine if the mechanism of the block of Kv1.5 ionic currents by external Ni2+ is similar to that for proton block. Ni2+ block is inhibited by increasing [K+]o, by mutating a histidine residue in the pore turret (H463Q) or by mutating a residue near the pore mouth (R487V) that is the homolog of Shaker T449. Aside from a slight rightward shift of the Q-V curve, Ni2+ had no effect on gating currents. We propose that, as with Ho+, Ni2+ binding to H463 facilitates an outer pore inactivation process that is antagonized by Ko+ and that requires R487. However, whereas Ho+ substantially accelerates inactivation of residual currents, Ni2+ is much less potent, indicating incomplete overlap of the profiles of these two metal ions. Analyses with Co2+ and Mn2+, together with previous results, indicate that for the first-row transition metals the rank order for the inhibition of Kv1.5 in 0 mM Ko+ is Zn2+ (KD approximately 0.07 mM) > or = Ni2+) (KD approximately 0.15 mM) > Co2+ (KD approximately 1.4 mM) > Mn2+ (KD > 10 mM).

Modulation of human ether-à-go-go-related K+ (HERG) channel inactivation by Cs+ and K+.

Unlike many other native and cloned K+ channels, human ether-à-go-go-related K+ (HERG) channels show significant Cs+ permeability with a PCs/PK (the permeability of Cs+ relative to that of K+) of 0.36 +/- 0.03 (n = 10). Here, we find that raising the concentration of external Cs+ (Cs+o) dramatically slows HERG channel inactivation without affecting activation. Replacement of 5 mM K+o by 135 mM Cs+o increased both inactivation and recovery time constants and shifted the mid-point of the steady-state inactivation curve by 25 mV in the depolarized direction (n = 6, P < 0.01). Raising [Cs+]o also modulated the voltage sensitivity of inactivation gating. With 130 mM Cs+i and 135 mM NMDG+o, the inactivation time constant decreased e-fold per 47.5 +/- 1.1 mV (n = 5), and when 20 mM Cs+ was added to the bath solution, the inactivation time constant decreased e-fold per 20.6 +/- 1.3 mV (n = 5, P < 0.01). A quantitative analysis suggests that Cs+o binds to a site in the pore that is influenced by the transmembrane electrical field, so that Cs+o-induced slowing of HERG inactivation is less prominent at strong depolarizations. K+o has effects that are similar to Cs+o and their effects were additive, suggesting Cs+o and K+o may share a common mechanism of action. The strong effects of Cs+ on inactivation but not on activation highlight the importance of ion and channel interactions during the onset of inactivation in the HERG channel.

Rapid induction of P/C-type inactivation is the mechanism for acid-induced K+ current inhibition.

Extracellular acidification is known to decrease the conductance of many voltage-gated potassium channels. In the present study, we investigated the mechanism of H(+)(o)-induced current inhibition by taking advantage of Na(+) permeation through inactivated channels. In hKv1.5, H(+)(o) inhibited open-state Na(+) current with a similar potency to K(+) current, but had little effect on the amplitude of inactivated-state Na(+) current. In support of inactivation as the mechanism for the current reduction, Na(+) current through noninactivating hKv1.5-R487V channels was not affected by [H(+)(o)]. At pH 6.4, channels were maximally inactivated as soon as sufficient time was given to allow activation, which suggested two possibilities for the mechanism of action of H(+)(o). These were that inactivation of channels in early closed states occurred while hyperpolarized during exposure to acid pH (closed-state inactivation) and/or inactivation from the open state was greatly accelerated at low pH. The absence of outward Na(+) currents but the maintained presence of slow Na(+) tail currents, combined with changes in the Na(+) tail current time course at pH 6.4, led us to favor the hypothesis that a reduction in the activation energy for the inactivation transition from the open state underlies the inhibition of hKv1.5 Na(+) current at low pH.

Molecular determinants of the inhibition of human Kv1.5 potassium currents by external protons and Zn(2+).

Using human Kv1.5 channels expressed in HEK293 cells we assessed the ability of H+o to mimic the previously reported action of Zn(2+) to inhibit macroscopic hKv1.5 currents, and using site-directed mutagenesis, we addressed the mechanistic basis for the inhibitory effects of H(+)(o) and Zn(2+). As with Zn(2+), H(+)(o) caused a concentration-dependent, K(+)(o)-sensitive and reversible reduction of the maximum conductance (g(max)). With zero, 5 and 140 mM K(+)(o) the pK(H) for this decrease of g(max) was 6.8, 6.2 and 6.0, respectively. The concentration dependence of the block relief caused by increasing [K(+)](o) was well fitted by a non-competitive interaction between H(+)(o) and K(+)(o), for which the K(D) for the K(+) binding site was 0.5-1.0 mM. Additionally, gating current analysis in the non-conducting mutant hKv1.5 W472F showed that changing from pH 7.4 to pH 5.4 did not affect Q(max) and that charge immobilization, presumed to be due to C-type inactivation, was preserved at pH 5.4. Inhibition of hKv1.5 currents by H+o or Zn(2+) was substantially reduced by a mutation either in the channel turret (H463Q) or near the pore mouth (R487V). In light of the requirement for R487, the homologue of Shaker T449, as well as the block-relieving action of K(+)(o), we propose that H(+) or Zn(2+) binding to histidine residues in the pore turret stabilizes a channel conformation that is most likely an inactivated state.