Block of CaV1.2 channels by Gd3+ reveals preopening transitions in the selectivity filter

Abstract

Using the lanthanide gadolinium (Gd(3+)) as a Ca(2+) replacing probe, we investigated the voltage dependence of pore blockage of Ca(V)1.2 channels. Gd(+3) reduces peak currents (tonic block) and accelerates decay of ionic current during depolarization (use-dependent block). Because diffusion of Gd(3+) at concentrations used (<1 microM) is much slower than activation of the channel, the tonic effect is likely to be due to the blockage that occurred in closed channels before depolarization. We found that the dose-response curves for the two blocking effects of Gd(3+) shifted in parallel for Ba(2+), Sr(2+), and Ca(2+) currents through the wild-type channel, and for Ca(2+) currents through the selectivity filter mutation EEQE that lowers the blocking potency of Gd(3+). The correlation indicates that Gd(3+) binding to the same site causes both tonic and use-dependent blocking effects. The apparent on-rate for the tonic block increases with the prepulse voltage in the range -60 to -45 mV, where significant gating current but no ionic current occurs. When plotted together against voltage, the on-rates of tonic block (-100 to -45 mV) and of use-dependent block (-40 to 40 mV) fall on a single sigmoid that parallels the voltage dependence of the gating charge. The on-rate of tonic block by Gd(3+) decreases with concentration of Ba(2+), indicating that the apparent affinity of the site to permeant ions is about 1 mM in closed channels. Therefore, we propose that at submicromolar concentrations, Gd(3+) binds at the entry to the selectivity locus and that the affinity of the site for permeant ions decreases during preopening transitions of the channel.

Figure 1.

Tonic and use-dependent effects of Gd³⁺ in different conditions, in which blocker potency is altered. Gray lines illustrate traces elicited in the presence of blocker scaled to the magnitude of control currents in the absence of blocker. (A) In 10 mM Ca²⁺, application of 0.2 μM Gd³⁺ reduced peak currents by about one half during the first pulse (I₁, tonic block), but to a much greater extent at the second pulse (I₂, use-dependent block). (B) With 10 mM Ba²⁺, both tonic and use-dependent block occurred at 25 nM Gd³⁺. (C) Although Ba²⁺ currents inactivated more rapidly in channels with the β₃ subunit rather than with the β_2a subunit (compare with B), tonic and use-dependent effects of Gd³⁺ were similar for both β subunits. (D) Exemplar Ca²⁺ currents through the EEQE mutant of the selectivity locus recorded with and without 2 μM Gd³⁺. (E) Dose–response curves for tonic effect of Gd³⁺ determined for different experimental conditions (indicated). Tonic block is described by relative magnitude of peak current during the first pulse. Before averaging, each peak current measured in the presence of blocker was normalized to its value in the absence of blocker. n = 6–8 cells were analyzed for every experimental condition. Lines are best fits by hyperbolic equation I = 1/(1 + [Gd³⁺]/IC₅₀), where IC₅₀ is the concentration of half-block. For Ba²⁺ currents, IC₅₀ = 0.03 ± 0.01 μM. Only one fit is shown for cells with β₃ and β_2a subunits, because the IC₅₀ was not significantly different in these cells. For Sr²⁺ currents, IC₅₀ = 0.12 ± 0.02 μM. For Ca²⁺ currents through the wild-type channel, IC₅₀ = 0.29 ± 0.05 μM. For Ca²⁺ currents trough the EEQE mutant in the selectivity locus, IC₅₀ = 2.1 ± 0.4 μM. (F) Dose–response curves for use-dependent blocking effects of Gd³⁺ determined for the same experimental conditions as in panel E. Use-dependent block is described by relative magnitude of peak current during the second pulse. Before averaging, the value of peak current at the second pulse was normalized to the value of peak current at the first pulse. Lines are best fits by hyperbolic equation I = A/(1 + [Gd³⁺]/IC₅₀), where A is a constant and IC₅₀ is the concentration of half-block. For Ba²⁺ currents in cells with the β₃ subunit, A = 0.57 ± 0.05, IC₅₀ = 0.022 ± 0.004 μM. For Ba²⁺ currents in cells with the β_2a subunit, A = 0.82 ± 0.03, IC₅₀ = 0.021 ± 0.004 μM. For Sr²⁺ currents, A = 0.64 ± 0.06, IC₅₀ = 0.08 ± 0.02 μM. For Ca²⁺ currents trough the wild-type channel, A = 0.61 ± 0.05, IC₅₀ = 0.15 ± 0.03 μM. For Ca²⁺ currents trough the EEQE mutant in the selectivity locus, A = 0.92 ± 0.07, IC₅₀ = 7.1 ± 2.6 μM.

Figure 2.

Voltage dependence of tonic and use-dependent block of Ba²⁺ currents as determined by the double-pulse experiments. Both components of Gd³⁺ block were relived at conditioning to large positive voltages. (A) Without Gd³⁺, test Ba²⁺ current was less after conditioning at 0 mV in comparison with the −60-mV conditioning. Application of 25 nM Gd³⁺ increased the effect of conditioning at 0 mV. However, test currents in the presence of Gd³⁺ increased after conditioning to 100 mV to the levels that exceeded maximal blocked currents and reached the magnitude of unblocked test current after prepulse to 100 mV. Therefore, both use-dependent and tonic block were relieved by prepulse to 100 mV. (B) Peak current–voltage relationships for the first pulse. Filled symbols plot values in unblocked channels. Open symbols are for currents in the presence of Gd³⁺. Before averaging (n = 6), in each cell peak currents at the first pulse were normalized to their maximal value in the absence of blocker. (C) Availabilities of current at the second pulse (test) plotted vs. voltage of the first pulse (conditioning). Before averaging, in each cell peak currents at the test pulse were normalized to their value determined with conditioning at −90 mV in the absence of blocker. Gd³⁺ did not significantly reduce Ba²⁺ currents measured after prepulses to voltages more positive than 70 mV. (D) Voltage dependence of tonic block determined from the data in B. (E) Voltage dependence of use-dependent block determined from the data in C.

Figure 3.

At any concentration of Gd³⁺, Ca²⁺ currents through the wild-type channel were minimal after conditioning at intermediate voltages. (A) Without Gd³⁺, test Ca²⁺ current was less after conditioning at 20 mV in comparison with the −60-mV conditioning. Application of 0.2 μM Gd³⁺ increased the effect of conditioning at 20 mV. However, the additional reduction of test current due to Gd³⁺ was partially relieved by conditioning at 100 mV. (B) Availabilities of test Ca²⁺ current plotted vs. conditioning voltage. Before averaging (n = 8), in each cell peak currents at the test pulse were normalized to their value determined with conditioning at −90 mV in the absence of blocker. In the absence of Gd³⁺ (circles), test currents were minimal after prepulses to 10 mV due to Ca²⁺-dependent inactivation. 0.1 μM Gd³⁺ (squares) enhanced the reduction of currents at intermediate voltages, but it did not significantly reduce Ca²⁺ currents measured after prepulses to voltages more positive than 90 mV. At greater amounts of Gd³⁺ (indicated), the voltage-dependent relief of block required stronger prepulses and it was not complete.

Figure 4.

Gd³⁺ block of Ca²⁺ currents through the channels with Ca²⁺-dependent inactivation diminished by coexpression with the mutant apo-calmodulin CAM1234. (A) Without Gd³⁺, conditioning to 20 mV had little effect on test Ca²⁺ current. At 0.2 μM Gd³⁺, conditioning at 20 mV caused significant reduction of Ca²⁺ current even though Ca²⁺-dependent inactivation was small. The reduction of test current due to Gd³⁺ was partially relieved by conditioning at 100 mV. (B) Availabilities of test Ca²⁺ current plotted vs. conditioning voltage. Before averaging (n = 5), in each cell peak currents at the test pulse were normalized to their value determined with conditioning at −90 mV in the absence of blocker. In the absence of Gd³⁺ (circles), the availability of test current was nearly a sigmoid function of voltage. With Gd³⁺, test currents were minimal after prepulses to intermediate voltages despite Ca²⁺-dependent inactivation was absent. With 0.1 μM Gd³⁺ (squares), the magnitude of Ca²⁺ currents measured after prepulses to voltages more positive than 80 mV was the same as without Gd³⁺. At greater amounts of Gd³⁺ (indicated), the voltage-dependent relief required stronger prepulses and it was not complete.

Figure 5.

Gd³⁺ block of Ca²⁺ currents through the EEQE mutant of the selectivity locus. The mutant diminishes Ca²⁺-dependent inactivation (Zong et al., 1994) and Gd³⁺ block (Fig. 1). (A) Without Gd³⁺, conditioning prepulses to different voltages (indicated) had little effect on test Ca²⁺ current. Addition of 2 μM Gd³⁺ blocked test currents by about one half regardless of the voltage of conditioning pulse (indicated). (B) Availabilities of test Ca²⁺ current plotted vs. conditioning voltage. Before averaging (n = 5), in each cell peak currents at the test pulse were normalized to their value determined with conditioning at −90 mV in the absence of blocker. In the absence of Gd³⁺ (filled circles), the availability of test current was nearly independent on voltage showing dramatic reduction of inactivation in the EEQE mutant. With 2 μM Gd³⁺ (open circles), the extent of additional block at intermediate voltages was small and there was no relief of block even at 150 mV.

Figure 6.

Reblock of closed channels. (A) Double-pulse protocol used to study kinetics of block of closed channels during interpulses of various duration (T_i) and voltage (V_i). (B) Prepulses to 100 mV had no measurable effect on test Ba²⁺ currents in the absence of Gd³⁺. (C) In presence of Gd³⁺, the magnitude of test current recorded briefly after the prepulse was similar to that without Gd³⁺. Test currents decreased with increasing interpulse duration. After long interpulses, test currents became equal to those without prepulse (tonic block).

Figure 7.

Dependence of block of closed channels on voltage. (A) Test currents were determined by pairs. First, a control current was recorded without prepulse (gray line). After 30 s at the holding potential of −90 mV, the pulse protocol shown by black line was applied. To avoid heavy-duty cycle effect, the next pair of currents was determined after holding the cell at −90 mV for at least 30 s. (B) Pairs of control (gray lines) and test Ba²⁺ currents (black lines) elicited without Gd³⁺ as described in A for the interpulse voltage of −60 mV and different interpulse duration (indicated). Test currents after 6 s interpulse were reduced by <10% because of inactivation at −60 mV. (C) Pairs of control and test Ba²⁺ currents elicited in the presence of 25 nM Gd³⁺ for the interpulse voltage of −60 mV. Control currents were reduced due to tonic block at the holding potential of −90 mV. Test currents recorded with a 50-ms interpulse were nearly as large as currents in the absence of Gd³⁺, but they decreased with the duration of the interpulse and became smaller than the control currents for interpulses longer than 2 s. (D) Averaged magnitudes of test currents determined at different interpulse voltage. n = 6–10 for each [Ba²⁺]. The lines are best fits by the sum of two exponential and a constant: I = ae^−bt + ce^−dt + e. For −90 mV interpulse, a = 0.84 ± 0.12, b = 1.89 ± 0.07 s⁻¹, c = 0, e = 1. For −60 mV, a = 0.86 ± 0.10, b = 1.91 ± 0.06 s⁻¹, c = 0.35 ± 0.12, d = 0.46 ± 0.08 s⁻¹, e = 0.55 ± 0.08. For −55 mV, a = 0.85 ± 0.09, b = 1.93 ± 0.07 s⁻¹, c = 0.39 ± 0.10, d = 0.43 ± 0.08 s⁻¹, e = 0.28 ± 0.07. For −50 mV, a = 0.95 ± 0.12, b = 2.01 ± 0.07 s⁻¹, c = 0.44 ± 0.10, d = 0.43 ± 0.09 s⁻¹, e = 0.17 ± 0.07. For −45 mV, a = 1.01 ± 0.11, b = 2.29 ± 0.08 s⁻¹, c = 0.51 ± 0.12, d = 0.44 ± 0.08 s⁻¹, e = 0.03 ± 0.03. (E) Estimates of rates of reblock determined from the fast kinetic component of reduction of test currents defined in D. k⁻ = b/(1 + E), k⁺ = bE/(1 + E), where b is the rate of the fast component and E = a/(a + c + e). The lines are drawn by eye.

Figure 8.

Dependence of block of closed channels on [Ba²⁺]. (A) Voltage pulse protocol to study kinetics of reblock at −90 mV. (B) Test currents obtained with interpulses of different duration (indicated). Thick lines show test currents obtained without prepulses. Gray lines show test currents without Gd³⁺ and black lines are traces obtained in the presence of 25 nM Gd³⁺. (C) Averaged magnitudes of test currents determined for different [Ba²⁺] (indicated). n = 5–8 for each [Ba²⁺]. The lines are best fits by the sum of an exponential and a constant: I = ae^−bt + c. For 2 mM, a = 1.04 ± 0.07, b = 5.94 ± 0.13 s⁻¹, c = 0.19 ± 0.06. For 10 mM, a = 0.86 ± 0.13, b = 1.83 ± 0.08 s⁻¹, c = 1. For 20 mM, a = 0.88 ± 0.12, b = 1.21 ± 0.07 s⁻¹, c = 1.23 ± 0.13. (D) Estimates of rates of reblock determined from the reduction of test currents defined in A. k⁻ = b/(1 + E), k⁺ = bE/(1 + E), where b is the rate of reduction of current and E = a/(a + c).

Figure 9.

Acceleration of Gd³⁺ block occurs at voltage steps that do not cause ionic currents. (A) Voltage pulse protocol. (B) Exemplar Ca²⁺ currents recorded in the presence of 1 μM Gd³⁺ with (black lines) and without (gray lines) the prepulse to 200 mV. Control currents (traces a and c) did not depend on the interpulse voltage V_i (indicated), but test currents recorded after the prepulse (traces b and d) decreased with V_i in the range where no ionic currents could be recorded (e.g., at −70 mV). At the same time, the voltage steps from −200 mV to V_i caused significant gating currents (indicated by arrows). (C) Averaged magnitude of test currents vs. voltage of the interpulse. Before averaging, each value was normalized to the magnitude at V_i = −100 mV determined in the same cell (n = 4).

Figure 10.

Prepulses used to study reblock of open channels changed neither magnitude nor kinetics of test currents in the absence of Gd³⁺. Exemplar currents recorded with (black lines) and without (gray lines) the prepulse to 200 mV. (A) Double-pulse protocol used to study kinetics of block of open channels during voltage steps (V) that activate ionic currents. (B) Prepulses to 200 mV had no measurable effect on test Ba²⁺ currents in the absence of Gd³⁺. The currents were recoded at V = 0 mV. (C) Prepulses to 200 mV had no measurable effect on test Ca²⁺ currents in the absence of Gd³⁺. The currents were recoded at V = 20 mV in a different cell.

Figure 11.

Kinetics of block of Ba²⁺ currents. (A) Test Ba²⁺ currents elicited by a pulse protocol similar to that in Fig. 10. Traces are shown for test voltages to −60–40 mV, increment 20 mV. Because of the prepulse to 200 mV, tonic block by Gd³⁺ was removed and reduction of currents during test voltage steps reflected reblock of open channels. (B) The averaged rates of best fits by the sum of an exponential and a constant to the decay phase of test currents recorded as in A are plotted vs. test voltage for different [Gd³⁺] (indicated). n = 5. (C) The averaged rates of current decay are plotted vs. [Gd³⁺] for different test voltages (indicated). The data are the same as in B.

Figure 12.

Kinetics of block of Ca²⁺ currents. (A) Test Ca²⁺ currents elicited similarly to that in Fig. 11. (B) The averaged rates of best fits by the sum of an exponential and a constant to the decay phase of test currents recorded as in A are plotted vs. test voltage for different [Gd³⁺] (indicated). n = 4. (C) The averaged rates of current decay are plotted vs. [Gd³⁺] for different test voltages (indicated). The data are the same as in B.

Figure 13.

The on-rates of Gd³⁺ block of “closed” channels (filled symbols) and “open” channels (open symbols) fall on a single sigmoid that parallels the voltage dependence of the gating charge movement.

Figure 14.

Gd³⁺ block does not change gating currents recorded at voltages before channel opening. Black traces, asymmetric currents (the sum of ionic and gating currents) in the bathing solution with 10 mM Ca²⁺; gray traces, asymmetric currents in the bathing solution with 10 mM Ca²⁺ and 1 μM Gd³⁺.