Steven J. Kehl

Molecular determinants of the inhibition of human Kv1.5 potassium currents by external protons and Zn2

Using human Kv1.5 channels expressed in HEK293 cells we assessed the ability of H+o to mimic the previously reported action of Zn2+ to inhibit macroscopic hKv1.5 currents, and using site‐directed mutagenesis, we addressed the mechanistic basis for the inhibitory effects of H+o and Zn2+. As with Zn2+, H+o caused a concentration‐dependent, K+o‐sensitive and reversible reduction of the maximum conductance (gmax). With zero, 5 and 140 mm K+o the pKH for this decrease of gmax 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 KD 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 Qmax 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 Zn2+ 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 Zn2+ binding to histidine residues in the pore turret stabilizes a channel conformation that is most likely an inactivated state.

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 α-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.

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+0) dramatically slows HERG channel inactivation without affecting activation. Replacement of 5 mm K+0 by 135 mm Cs+0 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 8mm Cs+1 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+0 binds to a site in the pore that is influenced by the transmembrane electrical field, so that Cs+0‐induced slowing of HERG inactivation is less prominent at strong depolarizations. K+0 has effects that are similar to Cs+0 and their effects were additive, suggesting Cs+0 and K+0 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.

Modulation of Kv1.5 Potassium Channel Gating by Extracellular Zinc

Zinc ions are known to induce a variable depolarizing shift of the ionic current half-activation potential and substantially slow the activation kinetics of most K(+) channels. In Kv1.5, Zn(2+) also reduces ionic current, and this is relieved by increasing the external K(+) or Cs(+) concentration. Here we have investigated the actions of Zn(2+) on the gating currents of Kv1.5 channels expressed in HEK cells. Zn(2+) shifted the midpoint of the charge-voltage (Q-V) curve substantially more (approximately 2 times) than it shifted the V(1/2) of the g-V curve, and this amounted to +60 mV at 1 mM Zn(2+). Both Q1 and Q2 activation charge components were similarly affected by Zn(2+), which indicated free access of Zn(2+) to channel closed states. The maximal charge movement was also reduced by 1 mM Zn(2+) by approximately 15%, from 1.6 +/- 0.5 to 1.4 +/- 0.47 pC (n = 4). Addition of external K(+) or Cs(+), which relieved the Zn(2+)-induced ionic current reduction, decreased the extent of the Zn(2+)-induced Q-V shift. In 135 mM extracellular Cs(+), 200 microM Zn(2+) reduced ionic current by only 8 +/- 1%, compared with 71% reduction in 0 mM extracellular Cs(+), and caused a comparable shift in both the g-V and Q-V relations (17.9 +/- 0.6 mV vs. 20.8 +/- 2.1 mV, n = 6). Our results confirm the presence of two independent binding sites involved in the Zn(2+) actions. Whereas binding to one site accounts for reduction of current and binding to the other site accounts for the gating shift in ionic current recordings, both sites contribute to the Zn(2+)-induced Q-V shift.

4‐Aminopyridine causes a voltage‐dependent block of the transient outward K+ current in rat melanotrophs.

1. Whole‐cell voltage‐clamp recordings were made from acutely dissociated melanotrophs obtained from adult rats. 2. In the presence of external Na+ and Ca2+ channel blockers and 20 mM‐tetraethylammonium (TEA) depolarizations to ‐40 mV or more evoked a fast‐activating fast‐inactivating outward K+ current (IK(f)). Double‐pulse experiments showed that steady‐state half‐inactivation occurred near ‐37 mV; half‐maximal activation of IK(f) occurred at ‐15 mV. Recovery from inactivation in most cells fitted a single exponential with a time constant of 40‐50 ms. 3. When applied either internally or externally, 1‐2.5 mM‐4‐aminopyridine (4‐AP) substantially reduced IK(f) but the degree of block was affected by the intensity, duration and frequency of depolarizing commands. 4. Analysis of the steady‐state voltage dependence of the block by 4‐AP showed that half‐maximal blocking occurred at approximately ‐31 mV. This implied that 4‐AP binds to the resting state of the IK(f) channel. 5. Studies of the time dependence for the blocking or unblocking of IK(f) showed that both processes were exponential with mean time constants of 1942 ms (at ‐70 mV) and 726 ms (at 20 mV), respectively. Recovery from inactivation was apparently unaffected by 4‐AP. 6. A four‐state sequential model in which 4‐AP reversibly binds to the resting state of the channel replicates the frequency dependence of the 4‐AP blockade.

Evidence for a bicuculline-insensitive long-lasting inhibition in the CA3 region of the rat hippocampal slice

Long-lasting inhibition (up to 2 s) of the commissurally-evoked response in the CA3 region of hippocampal slices was observed following a mossy fibre or commissural conditioning stimulus. Bicuculline applied iontophoretically or by superfusion (1-5 X 10(-6) M) blocked the early phase (20-40 ms) of the post-stimulus inhibition but either had no effect or potentiated the later inhibition.

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.

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 (τ 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.

External K+ relieves the block but not the gating shift caused by Zn2+ in human Kv1.5 potassium channels

1 We used the whole‐cell recording technique to examine the effect of extracellular Zn2+ on macroscopic currents due to Kv1.5 channels expressed in the human embryonic kidney cell line HEK293. 2 Fits of a Boltzmann function to tail current amplitudes showed that 1 mm Zn2+ shifted the half‐activation voltage from ‐10.2 ± 0.4 to 21.1 ± 0.7 mV and the slope factor increased from 6.8 ± 0.4 to 9.4 ± 0.7 mV. The maximum conductance in 1 mm Zn2+ and with 3.5 mm K+o was 33 ± 7 % of the control value. 3 In physiological saline the apparent KD for the Zn2+ block was 650 ± 24 μm and was voltage independent. A Hill coefficient of 1.0 ± 0.03 implied that block is mediated by the occupation of a single binding site. 4 Increasing the external concentration of K+ ([K+]o) inhibited the block by Zn2+. Estimates of the apparent KD of the Zn2+ block in 0, 5 and 135 mm K+ were 69, 650 and 2100 μm, respectively. External Cs+ relieved the Zn2+ block but was less effective than K+. Changing [K+]o did not affect the Zn2+‐induced gating shift. 5 A model of allosteric inhibition fitted to the relationship between the block by Zn2+ and the block relief by external K+ gave KD estimates of ≈70 μm for Zn2+ and ≈500 μm for K+. 6 We propose that the gating shift and the block caused by Zn2+ are mediated by two distinct sites and that the blocking site is located in the external mouth of the pore.

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.