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

Abstract

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.

FIGURE 1

Ba²⁺ blocks Kv1.5 in a concentration- and frequency-dependent manner. (A) With 0 mM pH_o 7.4 solution, 10-ms test pulses to +50 mV were applied at 1 Hz without (uppermost trace) and with 5 mM Ba²⁺. Shown are 65 consecutive traces from the time of Ba²⁺ application, which began immediately after the control trace. (Inset) The trace at the steady-state level of block was scaled 1.87× to match the peak amplitude of the control current. There was a small effect of Ba²⁺ on the onset of the K⁺ current. (B (i)) Trace 1 was recorded using a 10-ms pulse to +50 mV after a 1-min exposure to 1 mM Ba²⁺ in 0 mM at −80 mV. Traces 2–5 show the current from subsequent pulses applied at 1 Hz in the continued presence of Ba²⁺. Trace 25 shows the steady-state current in 1 mM Ba²⁺ with 1 Hz stimulation and Trace 100 is the steady-state current after recovery in 0 mM Ba²⁺. (B (ii)) Traces 1–5 and 25 are normalized with respect to Trace 100. There was a marked slowing of the rising phase of the current elicited by the first pulse after Ba²⁺ exposure, but this effect is diminished with subsequent pulses in the continued presence of Ba²⁺. (C) The time course of the onset and offset of the Ba²⁺ block. The average current amplitude in the last 1 ms of each pulse was normalized to the Ba²⁺-free control current. Down and up arrows indicate the time of fast Ba²⁺ application and withdrawal, respectively. Black circles (•) represent the normalized current amplitudes for the experiment shown in A. Single-exponential fits to the onset of and recovery from Ba²⁺ block give τ_block = 6.84 s and τ_unblock = 19.21 s, respectively. When the experiment was performed with the same stimulus protocol but with 20 mM Ba²⁺ (solid triangles), block onset was slightly faster (τ_block = 4.97 s) and the normalized steady-state current was smaller (0.37 vs. 0.50 for 5 mM Ba²⁺), but the time course of current recovery was the same. If the [Ba²⁺] was kept constant at 5 mM but the pulse frequency decreased to 0.2 Hz (open diamonds), the normalized steady-state level was also reduced (steady-state normalized current = 0.27). Although the onset of the block was not affected by the decrease in stimulation frequency, recovery with a 0.2-Hz pulse frequency was much slower (τ_unblock = 63.20 s) than that seen at 1 Hz. (D) The blocking rate (1/τ_block) is a nonlinear function of the [Ba²⁺]. τ_block values were assessed in 0 mM with 10-ms pulses to +50 mV applied at 1 Hz. The solid line represents the best fit of the data to a sequential two-site binding model (Eq. 1; see text for fit parameters). Data points are from a total of 46 cells, 3–8 cells/point. (E) The unblocking rate is linearly related to the pulse frequency. Values for τ_unblock were assessed with 0 mM and 20 mM using 10-ms pulses to +50 mV applied at 1, 0.7, 0.2, 0.066, or 0.033 Hz. The extrapolated off rate without pulsing (0 Hz) is 0.001 ± 0.002 s⁻¹. Data points are from a total of 53 cells, 5–24 cells/point.

FIGURE 2

Sequence alignment of hKv1.5 and Shaker from the C-terminal end of S5 to the end of the selectivity filter. The putative regions forming the turret, pore helix, and selectivity filter are shown.

FIGURE 3

The steady-state level of Ba²⁺ block is dependent on the stimulation frequency and the [Ba²⁺]. Test pulses to +50 mV for 10 ms were applied at either 30-s intervals (0.033 Hz (open circles)) or 1.43-s intervals (0.7 Hz (solid circles)) with 0 and a pH_o of 7.4. Ba²⁺ was applied at various concentrations using a computer-controlled fast perfusion system. The steady-state residual current (1 − proportion of blocked (P_B,SS)) at the end of the test pulse was normalized to the control test current before Ba²⁺ application and plotted against the concentration of Ba²⁺ applied (2 μM – 20 mM). The black solid lines represent the best fits of the two data sets to the Hill equation. Estimates of the apparent K_Ba,d and the Hill coefficient are 20.3 ± 2.6 μM and 1.12, respectively, for the data collected at 0.033 Hz, whereas the estimates from the 0.7 Hz data are 361 ± 151 μM and 1.16, respectively. Gray dashed lines represent the outcome of a simultaneous fit of the two data sets to Eq. 4. From the fit, k₂, K_Ba,s, k₋₂, and τ_d were estimated to be 0.15 s⁻¹, 0.8 mM, 0.0006 s⁻¹, and 59 ms, respectively (see text). The dotted gray line represents the solution for the residual normalized current (1 − P_r), where P_r is the proportion of channels blocked after a prolonged rest at the holding potential, i.e., at 0 Hz. A fit of 1 – P_r to the Hill equation gave a Hill coefficient of 1 and a K_Ba,d of 4 μM, indicating that the apparent K_Ba,d is profoundly affected by the pulse frequency. Data points are from a total of 29 cells (2–8 cells/point) for the 0.7 Hz data; and 45 cells (3–9 cells/point) for the 0.033 Hz data.

FIGURE 4

A model of the frequency-dependent Ba²⁺ binding replicates the time dependence of block onset and reversal. (A) A representative experiment in which a cell was given four 10-ms test pulses to +50 mV in 0 mM Ba²⁺ before a rapid changeover to 0.2 mM Ba²⁺ while test pulses continued to be applied at 0.1 Hz (open circles). After steady-state block was achieved, the pulse frequency was decreased to 0.033 Hz (open squares) and the block was allowed to reach a new steady state. The enhanced steady-state block observed with 0.033-Hz stimulus frequency was reversed upon reverting to 0.1-Hz pulses. The effects of changing the pulse frequency to 0.2 (○), 0.5 (Δ) and 0.7 (◊) Hz were subsequently examined using the same approach. Data points show current amplitudes normalized to the peak current at the end of the initial control test pulses. (B) Plot of simulated proportion of residual normalized current (1 – P_B,x) at the end of each test pulse shown in A, calculated using Eq. 5 and the values for k₂, K_Ba,s,, k₋₂, and τ_d derived from fits to the data of Fig. 3. The values for t_d, t_r, and τ_r were those derived from Fig. 3 (see text), whereas P_initial of Eq. 5 is 1 − the proportion of current at the end of the preceding pulse train. At each stimulus frequency, the numerical simulation provides a good estimate both of the steady-state level of block and the time course of the relaxation to that steady state.

FIGURE 5

Fast application of Ba²⁺ during a long depolarizing pulse reveals very weak open-channel block by 20 mM Ba²⁺. Current traces in response to depolarizing pulses to +50 mV for 10 s in 0 mM Ba²⁺-free solution (black trace) and with a rapid application and withdrawal of a 0 mM 20 mM Ba²⁺ solution during a subsequent pulse (gray trace). Down and up arrows indicate the start and end of the Ba²⁺ application, respectively. Exposure to Ba²⁺ caused a rapid fall in current amplitude of ∼10%. A similar effect was observed in six other cells.

FIGURE 6

Ba²⁺ unbinding from the open state is voltage-dependent and inhibited by increasing the [K⁺]_o. (A) Schematic of voltage-pulse and Ba²⁺ application protocol. A 1.5 s pulse to +50 mV was applied under 0.5 mM [K⁺]_o, pH_o 7.4 conditions. The cell was then exposed to 5 mM Ba²⁺ for 2 min, followed by a 4-min wash in control solution, after which the test pulse was repeated. (B) After Ba²⁺ exposure (gray trace), the current activation was much slower and well fitted by a single exponential (τ_act,app = 28.14 ms; compare to τ_act = 1.25 ms for the control response (black trace). (Inset) Current activation on an expanded timescale. These results are representative of those obtained from 11 cells. (C) Normalized currents after a 2-min exposure to 5 mM Ba²⁺ (with 0.5 mM ) recorded at two different test-pulse potentials. The activation time course at both voltages was well fitted by a single exponential. At +50 mV, τ_act,app was 22.56 ± 1.25 ms (n = 11), which was significantly faster (p < 0.001) than at +25 mV (τ_act,app = 65.59 ± 7.33 ms; n = 3). Each trace was normalized with respect to its own peak current. (D) Plot of the Ba²⁺ off rate (1/τ_act,app) derived from experiments such as those described in C, obtained between 0 and +100 mV in 25-mV increments. A fit of the data to the Woodhull model (see Methods) gave an estimate for δ_Ba,off of 0.51 ± 0.04. Data points are from a total of 21 cells; 3–11 cells/point. (E) After Ba²⁺ loading (in 0.5 mM ), the time course of the current onset in 0.5 mM or 10 mM was examined. The current activated significantly faster with 0.5 mM (τ_act,app = 22.56 ± 1.25 ms; n = 11) than with 10 mM (τ_act,app = 145.40 ± 6.15 ms; n = 5; p < 0.001). Traces were normalized as in C. (F) Plot of the Ba²⁺ off rate under varying [K⁺]_o at a test-pulse potential of +50 mV. The best fit of the data to the Hill equation (see Methods) gave a K_D for the inhibition of Ba²⁺ unbinding of 1.00 ± 0.08 mM. Data points are from a total of 31 cells, 5–11 cells/point.

FIGURE 7

Low pH_o inhibits both Ba²⁺ binding and unbinding from Kv1.5. (A) Diary plot of normalized current amplitudes at the end of 10-ms pulses to +50 mV applied at 1 Hz. Experiments were started in 0 mM pH 7.4 bath solution. Rapid switching to pH 5.5 solution caused a rapid fall of the current amplitude. Subsequent application of 20 mM Ba²⁺ (down arrow), still at pH 5.5, caused a further small decline. The rapid return (up arrow) to Ba²⁺-free, pH_o 7.4 solution resulted in a quick recovery of current amplitude (τ_recov = 3.1 s), which would not be expected had Ba²⁺ accessed the deep pore site. (B) In the converse of the experiment shown in A, 20 mM Ba²⁺ was applied (down arrow) at pH_o 7.4, resulting in 71% block of current, and was followed by a rapid wash-off of Ba²⁺ (up arrow) with pH 5.5 solution that caused an almost complete loss of current. Fast change of the bath solution back to pH 7.4 caused a rapid component of current recovery, followed by a large slow component (64% of total recovery) with a time constant of 23.6 s. These results are consistent with the interpretation that the proportion of Ba²⁺-loaded channels is not affected by high-frequency pulsing when the pH_o is low.