Voltage-dependent acceleration of Ca(v)1.2 channel current decay by (+)- and (-)-isradipine

Abstract

Inhibition of Ca(v)1.2 by antagonist 1,4 dihydropyridines (DHPs) is associated with a drug-induced acceleration of the calcium (Ca(2+)) channel current decay. This feature is contradictorily interpreted as open channel block or as drug-induced inactivation. To elucidate the underlying molecular mechanism we investigated the effects of (+)- and (-)-isradipine on Ca(v)1.2 inactivation gating at different membrane potentials. alpha(1)1.2 Constructs were expressed together with alpha(2)-delta- and beta(1a)- subunits in Xenopus oocytes and drug-induced changes in barium current (I(Ba)) kinetics analysed with the two microelectrode voltage clamp technique. To study isradipine effects on I(Ba) decay without contamination by intrinsic inactivation we expressed a mutant (V1504A) lacking fast voltage-dependent inactivation. At a subthreshold potential of -30 mV a 200-times higher concentration of (-)-isradipine was required to induce a comparable amount of inactivation as by (+)-isradipine. At +20 mV the two enantiomers were equally efficient in accelerating the I(Ba) decay. Faster recovery from (-)- than from (+)-isradipine-induced inactivation at -80 mV in a Ca(v)1.2 construct (tau((-)-isr.(Cav1.2))=0.74 s<tau((+)-isr.(Cav1.2))=2.85 s) and even more rapid recovery of V1504A (tau((-)-isr.(V1504A))=0.39 s<tau((+)-isr.(V1504A))=1.98 s) indicated that drug-induced determinants and determinants of intrinsic inactivation (V1504) stabilize the DHP-induced channel conformation in an additive manner. In the voltage range between -25 and 20 mV where the channels inactivate predominantly from the open state the (+)- and (-)-isradipine-induced acceleration of the I(Ba) decay in V1504A displayed similar voltage-dependence as intrinsic fast inactivation of Ca(v)1.2. Our data suggest that the isradipine-induced acceleration of the Ca(v)1.2 current decay reflects enhanced fast voltage-dependent inactivation and not open channel block.

Figure 1

Peak current inhibition and modulation of inactivation kinetics in α_1L/α₂-δ/β_1a channels by (+)- and (−)-isradipine. (A,B) Barium currents through α_1L/α₂-δ/β_1a channels during membrane depolarizations from −80 mV to 20 mV in control and in the presence of different concentrations of (+)- and (−)-isradipine (in μM). (C) Concentration-response relationships of peak I_Ba inhibition of α_1L/α₂-δ/β_1a channels by (+)- (dashed line, from Berjukow et al., 2000) and (−)-isradipine. Channel block was estimated as the ratio of peak current in the presence of the respective enantiomer compared to peak I_Ba in control. Data points represent the mean values from 4–11 experiments. The IC₅₀ and the Hill coefficient (n_H) for peak current block by (−)-isradipine were obtained by best fit of the data points to the general dose-response equation (see Methods) yielding: IC₅₀=34±8 μM, n_H=0.45±0.03, n=4. (D) Acceleration of I_Ba decay by (+)- and (−)-isradipine estimated as late current inhibition during a 3 s depolarizing test pulse from −80 to 20 mV. The inset shows three superimposed normalized I_Ba through α_1L/α₂-δ/β_1a in control and the presence of 1 μM (+)- and (−)-isradipine.

Figure 2

Drug-induced closed-state inactivation of α_1L/α₂-δ/β_1a channels at the subthreshold potential of −30 mV. (A) Effect of prepulses of variable duration from −80 mV to −30 mV on the peak current evoked by a subsequent test pulse to 20 mV (see inset). The smooth curves are biexponential functions fitted to the time course of mean I_Ba inactivation of α_1L/α₂-δ/β_1a channels (I_{Ba/normalised}=A_fast×exp(−τ/τ_fast)+A_slow×exp(−τ/τ_slow)+C). Asymptotic values are represented by the dotted lines. The parameters of the fit in control: A_fast=0.05, τ_fast=0.62 s, A_slow=0.06, τ_slow=13.0 s, C=0.89; in 1 μM (−)-isradipine: A_fast=0.15, τ_fast=0.58 s, A_slow=0.1, τ_slow=14.0 s, C=0.73; in 10 μM (−)-isradipine: A_fast=0.21, τ_fast=0.49 s, A_slow=0.22, τ_slow=11 s, C=0.56. The time course of the biexponential fit to 1 μM (+)-isradipine-induced inactivation (dashed line from Berjukow et al., 2000) is shown for comparison. (B) Drug-induced steady-state inactivation (dotted lines in A) induced by different concentrations of (+)- and (−)-isradipine is plotted as a function of the applied drug-concentration. Fitting of the data points to the dose-response equation yielded for (−)-isradipine: IC₅₀=13±5 μM, n_H=0.41±0.03 (n=4) and for (+)-isradipine: IC₅₀=72±5 nM, n_H=0.92±14 (n=3). (C) Kinetics of the fast component of drug-induced inactivation by (+)- and (−)-isradipine. The corresponding mean time constants (τ_fast) of the biexponential fits (shown in A) are indicated for different drug-concentrations. (D) The amplitude coefficient of the fast component (A_fast, see A) is illustrated for different drug concentration. (+)- (black bars) and (−)-isradipine- (hatched bars) induced fast inactivation at −30 mV are illustrated at different drug concentrations.

Figure 3

Inhibition of peak I_Ba and (+)- and (−)-isradipine-induced inactivation in mutant V1504A. (A) Concentration-response relationships of peak I_Ba inhibition of V1504A channels by (−)-isradipine. Data points represent the mean values from 3–5 experiments. The IC₅₀ and the Hill coefficient (n_H) for peak current block by (−)-isradipine were obtained by best fit of the data points to the dose-response equation yielding: IC₅₀=2.2±0.2 μM, n_H=0.53±0.03; The dashed line represents the fitted concentration response curve for channel block by (+)-isradipine (from Berjukow et al., 2000). (B) (+)- and (−)-isradipine accelerate I_Ba inactivation of V1504A. (+)- and (−)-induced effects on I_Ba decay were estimated as current inhibition during a 3 s depolarizing test pulse from −80 to 20 mV in per cent. Inset: Superimposed normalized I_Ba illustrating (+)- and (−)-isradipine-induced acceleration of the current decay compared to control.

Figure 4

Voltage-dependent acceleration of the I_Ba decay by (+)- and (−)-isradipine. (A) The time constants of I_Ba decay of V1504A and α_1L/α₂-δ/β_1a were estimated by fitting single (V1504A, control) or bi-exponential functions to the current decay. Currents were elicited by 30 s steps from a holding potential −80 mV to the indicated voltages. Open circles represent the time constant of V1504A inactivation in control, filled circles and squares represent the (+)- and (−)-isradipine induced current decay time constants in V1504A. Open diamonds show the fast voltage-dependent inactivation time constant of α_1L/α₂-δ/β_1a in control. (B) Representative I_Ba of the mutant V1504A elicited by 10 s depolarizing steps to −25 mV and 3 s steps to 20 mV from −80 mV in control (open circles) or the presence of 1 μM (+)-isradipine (filled circles).

Figure 5

Recovery of α_1L/α₂-δ/β_1a and V1504A channels from (+)- and (−)-isradipine-induced inactivation. Time course of I_Ba recovery from (−)-isradipine-induced inactivation after a 3 s conditioning prepulses to 20 mV (holding potential −80 mV). Test pulses to 20 mV were applied at various time intervals after the conditioning pulses. Peak I_Ba during the test pulses were normalized to peak I_Ba measured during the conditioning prepulse. Smooth curves in panels represent mono- or biexponential functions fitted to I_Ba recovery of α_1L/α₂-δ/β_1a (A) and V1504A (B) channels in control and in 1 μM (−)-isradipine. The parameters of fit were in (A) for control recovery (○): A=0.33, τ=0.61 s, C=1; recovery in 1 μM (−)-isradipine: A=0.63, τ=0.74 s, C=0.97. Broken line illustrates the corresponding time course of I_Ba recovery in 1 μM (+)-isradipine (fitted curve form Berjukow et al., 2000). (B) Recovery of V1504A in control (○): A_slow=0.11, τ_slow=13.4 s, C=0.98 and the presence of 1 μM (−)-isradipine: A_fast=0.42, τ_fast=0.39 s, A_slow=0.21, τ_slow=11.1 s, C=0.98. Dashed line illustrates recovery of I_Ba in 1 μM (+)-isradipine (data from Berjukow et al., 2000). (C) Time constant of recovery from fast inactivation in control (white column), 1 μM and 10 μM (−)-isradipine (hatched columns). Black columns illustrate the corresponding recovery from (+)-isradipine-induced inactivation (data from Berjukow et al., 2000). α_1L/α₂-δ/β_1a and V1504A channels recover significantly faster from (−)-isradipine-induced inactivation compared to (+)-isradipine. Note that the inactivation deficient mutant V1504A recovered faster from (+)- and (−)-isradipine-induced inactivation than α_1L/α₂-δ/β_1a channels (*P<0.01 compared to α_1L/α₂-δ/β_1a in control. #P<0.01 compared to recovery of the respective enantiomers in α_1L/α₂-δ/β_1a).

Figure 6

The two isradipine enantiomers differentially affect the stability of the drug-induced channel conformation. The scheme summarizes the principle findings of the present study. The left column illustrates the stronger tonic block of construct α_1L/α₂-δ/β_1a by (+)- compared to the (−)-enantiomer (illustrated by different fit of the two components in a schematic binding pocket). Superimposed currents in control (c), 1 μM (+)- and 1 μM (−)-isradipine (lower panel) are from Figure 1. The scheme on the right illustrates the lower stability of the (−)-isradipine-induced inactivated state in constructs α_1L/α₂-δ/β_1a and V1504A (Figure 5). The (−)-enantiomer is suggested to induce an inactivated state that is less stable than the channel conformation induced by (+)-isradipine. The experimental evidence for this hypothesis is illustrated in the lower panel summarizing the slower recovery of α_1L/α₂-δ/β_1a from (+)- than from (−)-isradipine-induced inactivation (data from Figure 5).