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

Abstract

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.

Figure 1. Simplified gating schemes describing the putative actions of H⁺ or Ni²⁺ ions on Kv1.5 channels at resting and depolarized potentials

First order rate constants are shown as k_xy, where x and y denote the states (identified by the subscripts 0, 1, 2 or 3) involved in the transition. The concentration of H⁺ or Ni²⁺ ions, also known as the ligand, is shown as [L]; L denotes a ligand-bound state. Scheme I: at rest (−80 mV) channels in the upper row are in the available (A or A-L) state and able to pass current during test depolarizations to +50 mV. Channels in the lower row are in the unavailable or resting inactivated state (U or U-L) and remain non-conductive during test depolarizations. Resting inactivation is defined as the transition at −80 mV from the A to U state or from the A-L to U-L state. See Methods for details. Scheme II: at +50 mV channels are either in the open (O or O-L) and conducting state or in the open-but-slow-inactivated (OI or OI-L) and non-conducting state. Slow inactivation is defined as the transition at +50 mV from the O to OI state, while H⁺- or Ni²⁺-enhanced slow inactivation is defined as the transition from the O-L to OI-L state. See Methods for details.

Figure 2. Effects of external H⁺ and Ni²⁺ on Kv1.5 current

In all panels, superimposed grey traces represent current recorded during 5 s voltage steps to +50 mV from −80 mV following prolonged exposure (20–40 s) to low pH or [Ni²⁺]. Black lines represent the fits of the current decay to a mono-exponential function with a time constant denoted by τ_inact. Each panel represents an experiment on a different cell. A, in 0 mm, low pH decreases both peak Kv1.5 current and τ_inact. Pooled data from 4 cells gave a pK_a of 6.9 and a Hill coefficient of 1.4. B, increasing [K⁺]_o antagonizes the effect of low pH on peak test current. In 3.5 mm the pK_a for the decrease in peak current was 6.2, with a Hill coefficient of 1.8 (n = 4 cells). C, Ni²⁺ also causes a concentration-dependent decrease in peak current but this is associated with relatively smaller decreases of the test current decay rate. From 4 cells, the fit of the [Ni²⁺] dependence of the mean normalized peak current amplitude to the Hill equation gave K_d and Hill coefficient values of 0.034 mm and 0.85, respectively. D, in 3.5 mm the K_d for the [Ni²⁺]-dependent decrease of peak current amplitude was 0.52 mm; the Hill coefficient was 1.2 (n = 4 cells). For reasons unclear to us, these K_d values for the Ni²⁺ effect are lower than those previously reported (Kwan et al. 2004).

Figure 3. The time course of enhanced slow inactivation and resting inactivation is pH dependent

A, superimposed current traces from the same cell showing the enhancement of current decay by low pH. The 0 mm K⁺ bathing solution was rapidly and transiently switched from pH 7.4 to low pH during a 20 s step from −80 mV to +50 mV. Dashed lines represent mono-exponential fits of the current decay with τ_inact values of 4.88 s, 1.11 s and 0.25 s at pH 7.4, 6.4 and 5.9, respectively. B, current trace showing the onset of resting inactivation induced at pH 5.4 in 0 mm. After a 20 ms control pulse to +50 mV at pH 7.4, the external pH was decreased and a pulse train with increasing interpulse intervals was applied during a single sweep. C, resting inactivation was also assessed with multiple (superimposed) sweeps. In each sweep a single test pulse was applied at a known interval after the switch to pH 5.4; the interval was increased for each successive sweep. The final, control, trace was obtained without prior exposure to low pH. D, the onset of resting inactivation induced by pH 5.4 in 3.5 mm was measured as described for B. E, test currents from (B–D) were normalized to their respective controls and plotted against the cumulative time spent at −80 mV at pH 5.4. Inset, the same plot on a longer time scale shows the steady-state level. The continuous and dashed lines represent mono- and bi-exponential fits of the data, respectively; τ_RI at pH 5.4 in 0 was 171 ms and 122 ms measured with a train and single pulses, respectively. In 3.5 mm at pH 5.4, τ_RI,fast and τ_RI,slow were 131 ms and 1.24 s, respectively. F, time constants for low pH enhanced slow inactivation and resting inactivation derived from experiments such as those in A and B are plotted against pH. All data points represent the mean ±s.e.m. from at least 3 cells.

Figure 4. The time course of enhanced slow inactivation and resting inactivation is also [Ni²⁺] dependent

A, superimposed current traces from the same cell showing the enhancement of current decay by external Ni²⁺. During a 20 s step from −80 mV to +50 mV, the control (pH 7.4, 0 mm K⁺) bath solution was rapidly and transiently switched to one containing Ni²⁺. Dashed lines represent mono-exponential fits of the current decay with τ_inact values of 4.08 s, 1.65 s and 0.34 s in 0, 0.1, and 10 mm Ni²⁺, respectively. B, current trace showing the onset of Ni²⁺-induced resting inactivation in 0 mm. After a 20 ms control pulse from −80 mV to +50 mV in 0 mm Ni²⁺, resting inactivation was assessed by switching to 10 mm Ni²⁺ solution and applying a pulse train with increasing interpulse intervals. C, the experimental protocol in B was repeated, albeit on a different time scale, with 3.5 mm. D, test currents from (B and C) were normalized to their respective controls and plotted against the cumulative time spent at −80 mV in Ni²⁺. For comparison, results from an experiment using 0.05 mm Ni²⁺ are also shown. Continuous and dashed lines represent mono- and bi-exponential fits of the data, respectively. E, time constants for Ni²⁺-enhanced current decay and resting inactivation derived from experiments such as those in A and B are plotted against [Ni²⁺]. Data points represent the mean ±s.e.m. of 3–7 cells.

Figure 5. The kinetics of recovery from H⁺- or Ni²⁺-enhanced slow inactivation or resting inactivation

A, current traces recorded in 0 mm from the same cell showing: recovery at pH 7.4 from control slow inactivation (□); recovery at pH 7.4 from slow inactivation enhanced by pH 5.4 (•); and recovery at pH 7.4 from resting inactivation induced by pH 5.4 (▵). The voltage clamp protocol consisted of a 5 s step to +50 mV, except for the resting inactivation trace, followed by a train of 20 ms test pulses to +50 mV. The dotted horizontal lines indicate the duration of the exposure to pH 5.4 solution. B, peak test currents from A, normalized with respect to the peak control current, are plotted against the cumulative recovery time spent at the −80 mV holding potential. The continuous lines represent the simultaneous fit to a double exponential function of the time course of recovery from control slow inactivation (□), H⁺-enhanced slow inactivation (•) and resting inactivation (▵). The resulting values for τ_rec,f and τ_rec,s were 1.48 s and 9.14 s, respectively. C, normalized peak recovery currents measured with the same protocol as in (A), but with 4 mm Ni²⁺ instead of low pH, are plotted against the cumulative recovery time. The recovery from control (□) and Ni²⁺-enhanced (•) slow inactivation were simultaneously fitted to a double exponential function with τ_rec,f = 0.59 s and τ_rec,s = 10.6 s. Recovery from Ni²⁺-enhanced slow inactivation was entirely via the slow phase. The inset graph shows that recovery from Ni²⁺-induced resting inactivation (▵) was faster than that following enhanced slow inactivation. The continuous lines represent separate fits of each data set to a mono-exponential function. τ_rec was 10.8 s for enhanced slow inactivation and 6.7 s for resting inactivation.

Figure 6. Current recovery during a test depolarizing pulse occurs following either H⁺- or Ni²⁺-enhanced slow inactivation or resting inactivation

The experimental approach was similar to that used in Fig. 5 except that the test pulse duration was longer and multiple test sweeps were used. An intersweep interval of 60 s allowed for full recovery between sweeps. For the top panels, after a 20 ms control pulse from −80 mV to +50 mV in pH 7.4, 0 mm solution, resting inactivation at −80 mV was induced by a 10 s application of either pH 6.4 (Aa) or 2 mm Ni²⁺ (Ba) solution. To monitor recovery, the test solution was replaced by control solution and the cell held at −80 mV for varying intervals before a 5 s test pulse was applied. For the lower panels, recovery from slow inactivation enhanced by pH 6.4 (Ab) or 2 mm Ni²⁺ (Bb) was assessed in the same way, except that low pH and Ni²⁺ were applied 100 ms after the beginning of a 5 s pulse from −80 mV to +50 mV. For each column, traces were recorded from the same cell. These results show that with either ligand and following either resting inactivation (top panels) or enhanced slow inactivation (lower panels, and see also Figs 3A and 4A) there can be the recovery of current during a depolarizing pulse and that the kinetics of that recovery are qualitatively similar for a given ligand. Dotted lines represent single exponential fits of the amplitude of the fast phase of the test current (○). As shown in Fig. 5, recovery at −80 mV following low pH exposure is faster than that following Ni²⁺ exposure.

Figure 7. The time course for recovery from 0 -induced resting inactivation is the same as that for recovery from enhanced slow inactivation

Using cells expressing Kv1.5 channels at high density, current recordings were made in pH 5.4, 3.5 mm K⁺ solution. Following a 20 ms control pulse, the recovery of current in 3.5 mm was monitored, in A, after a 500 ms pulse to +50 mV or, in B, after channel availability was decreased by a 5 s exposure to K⁺-free, pH 5.4 solution. Recovery was monitored using 20 ms test pulses applied at increasing intervals within the same sweep. C, peak test current amplitudes from A and B were normalized with respect to the initial control pulse and plotted against the cumulative recovery time spent at −80 mV. Both data sets were well fitted by a mono-exponential function and had similar time constants. Data shown are from the same cell and are representative of 3 experiments. D, data from another cell (representative of 4 such experiments), where the experiment protocol was analogous to that of panels A and B, and was performed at pH 7.4 with 2 mm Ni²⁺. As with low pH, the time courses of recovery from enhanced slow inactivation and resting inactivation were similar.

Figure 8. Theoretical outcomes of the modulation of slow inactivation by ligand binding

A, the gating model has several closed states in the activation pathway and the open state (O) is coupled to two slow inactivation states, I_f and I_s. For simplicity, the ligand binding/unbinding steps of Scheme II (Fig. 1) are omitted from this revised gating scheme. Although the inactivation rate constants (O → I_f, I_f→ O, O → I_s and I_s→ O of 0.5, 2.2, 0.2 and 0.1 s⁻¹) have been chosen to give macroscopic current behaviour similar to that in Kv1.5 during a 5 s pulse to +50 mV, the simulation is provided for the purpose of illustration only. B, the outcome of the model assuming that ligand application, represented by the bar above the superimposed traces, makes one or both of the inactivation states absorbing (I → O = 0 s⁻¹) but affects neither of the O → I transitions. The relatively modest effect of making I_s (dotted trace), I_f (short dashes trace) or both I_f and I_s (long dashes trace) absorbing emphasizes that rapid current decay, such as that observed in Kv1.5 with H⁺, must involve an increase of the rate constant for the O → I transition. C, the selective enhancement of the O → I_f transition during a 2 s ligand application produces a rapid, mono-exponential decay of current that is followed by a rapid recovery that ‘overshoots’ the control trace. D, ligand application that selectively enhances the O → I_s transition is followed by a slowly recovering current. E, in the model, which does not incorporate inactivation from the resting state, an increase of the O → I_f rate constant and a decrease of the I_f→ O rate constant, which mimics the time course and extent of the current decay in pH 5.4 solution (Fig. 3), causes only a 12% decrease of peak current compared to the control response. This outcome supports the conclusion that the enhancement of slow inactivation makes, at best, a minor contribution to the reduction of peak current observed with Ni²⁺ or H⁺ (e.g. Fig. 2). The calibration bar represents 1 s for panels B–D and 0.1 s for panel E.

Figure 9. Numerical simulation of resting inactivation caused by Ni²⁺ or low pH

For the simulation with Ni²⁺, the rate constants, based on the scheme in panel F, were: k₀₁ = 110 mm⁻¹ s⁻¹; k₁₀ = 150 s⁻¹; k₁₂ = 1.422 s⁻¹; k₂₁ = 0.036 s⁻¹; k₂₃ = 2.19 s⁻¹; k₃₂ = 1100 mm⁻¹ s⁻¹; k₀₃ = 0.008 s⁻¹; k₃₀ = 0.14 s⁻¹. A, the time dependence of the Ni²⁺-induced decrease of availability, measured as the sum of the proportions of channels in states A and ANi²⁺, was well-fitted by a single exponential. The [Ni²⁺] for the top sweep was 2.5 μm and was doubled for each subsequent sweep. B, the steady-state availability (□), measured from the traces of panel A, is plotted against the [Ni²⁺]. The dashed curve is the fit of the simulated data to the Hill equation (K_d = 36 μm; n_H = 1.0) and the continuous curve is the fit to the experimental data (K_d = 33 μm; n_H = 1.15). C, the values for τ_RI for the simulated (□, from fits of selected traces in A) and experimental (▪) data are plotted against the [Ni²⁺]. For the simulation of extracellular pH induced resting inactivation (D and E), protonation was assumed to trigger an inactivation process different from that induced by Ni²⁺ binding. For ○ in D and E, the rate constants were: k₀₁ = 2 × 10⁹m⁻¹ s⁻¹; k₁₀ = 9000 s⁻¹; k₁₂ = 4.356 s⁻¹; k₂₁ = 0.176 s⁻¹; k₂₃ = 182.3 s⁻¹; k₃₂ = 2 × 10¹⁰m⁻¹ s⁻¹; k₀₃ = 0.03 s⁻¹; k₃₀ = 0.6 s⁻¹ and the binding was assumed to involve a single site (n_H = 1). For □ in D and E, the steepness of the experimental availability curve (n_H = 1.5) was mimicked by changing k₀₁ and k₃₂ to 5 × 10¹²m^−1.5 s⁻¹ and 5 × 10¹³m^−1.5 s⁻¹ and multiplying k₀₁ and k₃₂ by [H⁺]^1.5. The control pH was 7.4 and the test pH ranged from 7.2 to 5.4 in 0.2 pH unit steps. The best fits of the simulated availability data for ○ and □ to the Hill equation are indicated by the dotted line (K_d = 1.8 × 10⁻⁷m; pK_a = 6.73, n_H = 1.0) and the dashed line (K_d = 1.8 × 10⁻⁷m, pK_a = 6.73, n_H = 1.5), respectively. The continuous line is the fit of the experimental concentration dependence (K_d 1.6 × 10⁻⁷m, pK_a = 6.8, n_H = 1.5) taken from Kehl et al. (2002). In panel E the time constants for the loss of availability for the two simulations described for panel D is compared to the experimental values taken from Fig. 3F and represented by ▪. F, the state diagram for resting inactivation at −80 mV. See Fig. 1A for explanation.