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(Circulation Research. 2001;89:700.)
© 2001 American Heart Association, Inc.

Cellular Biology

On the Role of Ca²⁺- and Voltage-Dependent Inactivation in Ca_v1.2 Sensitivity for the Phenylalkylamine (-)Gallopamil

Stanislav Sokolov, Eugen Timin, Steffen Hering

From the Institut für Biochemische Pharmakologie (S.S., S.H.), Innsbruck, Austria; and A.V. Vishnevsky Institute of Surgery (E.T.), Moscow, Russia.

Correspondence to Steffen Hering, Institut für Biochemische Pharmakologie, Peter-Mayr-Straße 1, A-6020 Innsbruck, Austria. E-mail Steffen.Hering.{at}uibk.ac.at

	Abstract

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Abstract— L-type calcium channels (Ca_v1.m) inactivate in response to elevation of intracellular Ca²⁺ (Ca²⁺-dependent inactivation) and additionally by conformational changes induced by membrane depolarization (fast and slow voltage-dependent inactivation). Molecular determinants of inactivation play an essential role in channel inhibition by phenylalkylamines (PAAs). The relative impacts, however, of Ca²⁺-dependent and voltage-dependent inactivation in Ca_v1.2 sensitivity for PAAs remain unknown. In order to analyze the role of the different inactivation processes, we expressed Ca_v1.2 constructs composed of different ß-subunits (ß_1a-, ß_2a-, or ß₃-subunit) in Xenopus oocytes and estimated their (-)gallopamil sensitivity by means of the two-microelectrode voltage clamp with either Ba²⁺ or Ca²⁺ as charge carrier. Ca_v1.2 consisting of the ß_2a-subunit displayed the slowest inactivation and the lowest apparent sensitivity for the PAA (-)gallopamil. A significantly higher apparent (-)gallopamil-sensitivity with Ca²⁺ as charge carrier was observed for all 3 ß-subunit compositions. The kinetics of Ca²⁺-dependent inactivation and slow voltage-dependent inactivation were not affected by drug. The higher sensitivity of the Ca_v1.2 channels for (-)gallopamil with Ca²⁺ as charge carrier results from slower recovery (_rec,Ca 15 seconds versus _rec,Ba 3 to 5 seconds) from a PAA-induced channel conformation. We propose a model where (-)gallopamil promotes a fast voltage-dependent component in Ca_v1.2 inactivation. The model reproduces the higher drug sensitivity in Ca²⁺ as well as the lower sensitivity of slowly inactivating Ca_v1.2 composed of the ß_2a-subunit.

Key Words: Ca_v1.2 • ß-subunit • Ca²⁺-dependent inactivation • voltage-dependent inactivation • PAA (-)gallopamil

	Introduction

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L-type calcium (Ca²⁺) channels (Ca_v1.m, see Ertel et al¹ for nomenclature) are the prime targets of the three clinically relevant classes of Ca²⁺ antagonists, ie, phenylalkylamines (PAAs), benzothiazepines (ie, diltiazem) and 1,4-dihydropyridines (DHPs).^2–5

About 20 years ago, several groups demonstrated that Ca_v1.m inhibition by PAAs and diltiazem occurs predominately during membrane depolarizations ("use-dependent" action) when the channels are primarily in the open or inactivated conformation.^2–4,6 Since then, numerous studies in myocardial and smooth muscle cells have shown that Ca²⁺ channel inhibition by these compounds, as well as by the new compound mibefradil, reflect a balance between channel shut-off during membrane depolarization and subsequent recovery at rest.^7–11

Over the last three years there has been substantial progress toward understanding the role of inactivation in Ca²⁺ channel inhibition by Ca²⁺ antagonists. Site directed mutagenesis enabled a detailed analysis of the impact of this process in PAA sensitivity. A comparison of the PAA sensitivities of Ca_v2.1 mutants and chimeric channel constructs inactivating at different rates revealed a strong correlation between the rates of fast voltage-dependent inactivation and apparent PAA sensitivities.^11–13 Similar findings were reported for Ca²⁺ channel block by diltiazem.^11,14 Subsequently, the studies concentrated on the localization of inactivation determinants that are relevant for channel inhibition. Thus, inactivation determinants in the loop between domains I and II and ß-subunit interaction have been shown to control PAA sensitivity of a Ca_v2.1 mutant¹³ as well as the mibefradil-sensitivity of wild type Ca_v2.1.¹⁵

The underlying mechanism in voltage-dependent inactivation modulation of Ca_v1.2 inhibition by PAAs remains poorly understood. In the present study, we undertake a systematic analysis of the role of fast voltage-dependent (ß-subunit–modulated), slow voltage-dependent, and additional Ca²⁺-dependent inactivation in PAA sensitivity of Ca_v1.2. We show here that the ß-subunit-dependent inactivation of Ca_v1.2 affects the channel inhibition by (-)gallopamil. Furthermore, the apparent PAA sensitivity of Ca_v1.2 channels is dependent on the charge carrying ion. A significantly stronger use-dependent inhibition with Ca²⁺ than with Ba²⁺ as charge carrier for various ß-subunit combinations was observed. Slow voltage-dependent inactivation was not affected by drug. By means of a mathematical model, we have simulated (-)gallopamil action on Ca_v1.2. Our data are consistent with the hypothesis that the drug promotes fast voltage-dependent inactivation.

	Materials and Methods

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Expression of the Ca_v1.2 Construct
A Ca_v1.2-deduced Ca²⁺ channel construct (named herein _1L) used in the present study corresponds to _1C-a cDNA¹⁶ with part of the amino terminus replaced by carp _1S-sequence. This substitution results in a considerable increase of the expression density (see Grabner et al¹⁷ for details). We observed no significant differences in PAA sensitivity of Ca²⁺ channels consisting either of _1L- or _1C-a-subunits (data not shown). The _1L-subunit was coexpressed with either ß_1a(M25514), ß_2a(M80545), or ß₃(M88751) subunits. The constructs were inserted into the polyadenylating transcription plasmid pSPCBI-2.

Electrophysiology
Two-microelectrode voltage clamp of Xenopus oocytes was performed 2 to 7 days after microinjection of approximately equimolar mixtures of cRNAs: _1L-(0.3 ng/50 nL), ß_1a/ß_2a/ß₃-(0.1 ng/50 nL) and ₂--subunits (0.2 ng/50 nL), as previously described.¹⁷ All experiments were performed at room temperature in either 10 mmol/L Ba(OH)₂, 95 mmol/L NaOH, 2 mmol/L CsOH, 5 mmol/L HEPES (called herein 10 mmol/L Ba²⁺ solution), or 10 mmol/L Ca(OH)₂, 95 mmol/L NaOH, 2 mmol/L CsOH, 5 mmol/L HEPES (10 mmol/L Ca²⁺ solution). pH was adjusted to 7.4 with methanesulfonic acid.

Activation of endogenous Ca²⁺-activated Cl^--conductance was eliminated by injecting 25 to 50 nL of 0.1 mol/L BAPTA solution into the oocytes 30 to 60 minutes before the experiments. Leakage current correction was performed by using average values of scaled currents elicited by a 10 mV hyperpolarizing voltage step.

The potentials of maximal activation of _1L/₂-/ß_1a(ß_2a,ß₃) channels ranged between 0 and 10 mV in 10 mmol/L Ba²⁺ solution and between 10 and 20 mV in 10 mmol/L Ca²⁺ solution.

Drug-sensitivity was estimated as use-dependent peak current inhibition during 10 test pulses (1 second) applied at 0.5 Hz from -80 mV to the peak potential of the corresponding current-voltage relationship. Use-dependent inhibition was measured after a 3 minutes equilibration of the oocytes in the drug-containing solution. To estimate the accumulation of Ca²⁺ channels in inactivation under control conditions, similar pulse trains were applied in the absence of drug.

To accurately estimate Ca²⁺-dependent and voltage-dependent components in _1L/ß_2a current decay, a protocol with flexible sampling interval was used (see Figure 4A). During the first 400 ms of current decay, data points were sampled with 1 kHz, allowing a high resolution of the Ca²⁺-dependent component. The subsequent part of the 40-second-long pulse was sampled at 20 Hz.

View larger version (20K): [in this window] [in a new window]   Figure 4. Effects of (-)gallopamil on voltage- and Ca2+-dependent kinetics of 1L/ß2a current decay. A and B, Normalized ICa (at 0 mV) and IBa (at +10 mV) elicited by 40-second depolarisations under control conditions and in the presence of 50 µmol/L (-)gallopamil. A pulse protocol with flexible sampling rate (see Materials and Methods) enabled an accurate estimation of the fast Ca2+-dependent component (A, inset). C, (-)Gallopamil accelerates the slow, voltage-dependent component of the ICa decay in concentration-dependent manner. Control (squares), 10 µmol/L (triangles), and 50 µmol/L (circles). Inset demonstrates that U-shaped Ca2+-dependent component of current decay is not accelerated by the drug. D, Effect of (-)gallopamil on the voltage-dependent IBa inactivation (same designations as in C). Control Ba values are shown in the inset. Note the similar voltage-dependence of the drug-induced current decay and intrinsic voltage-dependent inactivation.

Recovery from fast voltage-dependent inactivation and inhibition by PAA was studied at a holding potential of -80 mV after depolarizing Ca²⁺ channels during a 3-second prepulse to 10 mV (in 10 Ba²⁺ solution) or 20 mV (in 10 Ca²⁺ solution) by applying 30-ms test pulses at various time intervals after the conditioning prepulse. Peak current values were normalized to the peak current measured during the prepulse. The time course of I_Ba or I_Ca recovery was fitted to a mono- or double-exponential function (I_{Ba/Ca,recovery}=A · exp(-t/_fast)+B · exp(-t/_slow)+C).

Recovery from slow voltage-dependent inactivation was studied by a similar double-pulse protocol, with the 30-ms test pulses applied at 10, 20, 30, 60, 90, 120, and 180 seconds after the 30-second conditioning pulse to +10 mV (in Ba²⁺). Peak I_Ba during recovery was normalized to peak of the last test pulse. The data were fitted with a single-exponential function. All data are given as mean±SEM, if not specified, n4. Statistical significance was calculated according to Student’s unpaired t test (P<0.05).

	Results

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ß-Subunit Composition of Ca_v1.2 Affects Sensitivity for (-)Gallopamil
Coexpressing the Ca_v1.2 construct (_1L) with the ß_2a-subunit (corresponding channels named herein _1L/ß_2a) induced a slow rate of voltage-dependent inactivation of I_Ba, whereas coexpression of the ß_1a- and ß₃-isoforms (_1L/ß_1a and _1L/ß₃ channels) promoted a more rapid time course of inactivation (Figure 1A, see also Cens et al¹⁸). In order to quantify the inactivation kinetics of the different constructs, we used the coefficient r₁₀₀₀ (ie, the current remaining after 1000 ms normalized to the peak current, Figure 1B).

View larger version (26K): [in this window] [in a new window]   Figure 1. Inactivation kinetics of CaV1.2 composed of the 1L, 2-, and different ß-subunits. Normalized representative currents elicited by 3-second steps from -80 mV to +10 mV in 10 mmol/L Ba2+ solution (A) and to +20 mV in 10 mmol/L Ca2+ solution (C). The different inactivation rates in Ba2+ (B) and Ca2+ (D) are expressed by the coefficient r1000 (fraction of noninactivated current during a 1-second step with respect to peak current).

Ca²⁺-dependent inactivation caused an additional rapid component in the current decay in all constructs (Figure 1C). Irrespective of the charge carrier, _1L/ß_2a-channels inactivated considerably slower than _1L/ß_1a- and _1L/ß₃-channels (Figures 1B and 1D).

PAA sensitivity was estimated as use-dependent peak current inhibition during 10 test pulses applied at 0.5 Hz (Figure 2, see Materials and Methods for details). (-)Gallopamil (10 µmol/L) inhibited I_Ba through _1L/ß_2a less efficiently (34±2%, n=10) than through _1L/ß_1a (54±5%, n=6) and _1L/ß₃ (62±2%, n=5) channels (Figure 2B). This finding is in accordance with our previous observations on PAA-sensitive Ca_v2.1 mutants.¹³

View larger version (29K): [in this window] [in a new window]   Figure 2. The apparent (-)gallopamil sensitivity of Cav1.2 construct is dependent on the ß-subunit composition and the charge carrying ion. A, Representative currents elicited from a holding potential of -80 mV by 10 (1-second) pulses applied at 0.5 Hz in the presence of 10 µmol/L (-)gallopamil in 10 mmol/L Ba2+ (left panels) and 10 mmol/L Ca2+ solution (right panels). Depolarizing voltages correspond to the peak current potential of IBa (+10 mV) and ICa (+20 mV). B, The apparent (-)gallopamil-sensitivity of a given construct is estimated as peak current inhibition during 10 depolarizing pulses in percentage. The white bars represent the accumulation of channels in intrinsic inactivation in the absence of drug (C, control); the black and hatched bars show the current inhibition by 10 µmol/L (-)gallopamil in Ba2+ and Ca2+ solutions, respectively.

With 10 mmol/L Ca²⁺ as charge carrier, a significantly stronger peak current inhibition for all Ca_v1.2 constructs (_1L/ß_1a: 69±4%; _1L/ß_2a: 54±3%; _1L/ß₃: 71±2%; n6, Figure 2) was observed. The effect was most prominent for _1L/ß_2a channels exhibiting in 10 mmol/L Ca²⁺ solution a remarkably higher PAA sensitivity than in 10 mmol/L Ba²⁺. The order of PAA sensitivity was found to be similar for both ionic conditions (ie, _1L/ß_31L/ß_1a>_1L/ß_2a).

Role of Ca²⁺- and Voltage-Dependent Inactivation in Ca_v1.2 Inhibition by (-)Gallopamil
It is well-established that Ca_v1.2 inactivates in a Ca²⁺- and voltage-dependent manner.^19–21 Figure 3A illustrates representative I_Ca through _1L/ß₃ during 3-second depolarizing steps to different potentials. The rapid Ca²⁺-dependent component in the current decay was absent if Ba²⁺ served as charge carrier (compare Figures 1A and 1C). Moreover, voltage-dependence of the fast component of current decay possesses a characteristic U-shape of a Ca²⁺-dependent process (Figure 3B, _Ca,+30mV is about 4 times slower than _Ca,0mV).^20,22,23,24 A second slow component of the current decay was similar in Ba²⁺ and Ca²⁺ solutions (Figures 1A and 1C). This slow component was monotonically dependent on membrane potential suggesting a voltage-dependent inactivation mechanism (Figure 3C).

View larger version (12K): [in this window] [in a new window]   Figure 3. Ca2+- and voltage-dependent inactivation of 1L/ß3 channels. A, Representative ICa during 3-second test pulses from -80 mV to the indicated voltages. The time constants of the Ca2+-dependent (B, squares, n=12) and voltage-dependent (C, triangles, n=12) components were estimated by fitting the current decay by a double-exponential function. The U-shaped dependence on voltage is observed only for the fast component (Ca).

The different kinetics of Ca²⁺- and voltage-dependent inactivation enabled us to study (-)gallopamil effects on both processes individually. Because of their slow rate of voltage-dependent inactivation, _1L/ß_2a channels are particularly suitable for this analysis (see Figure 4A and inset for representative currents at 0 mV where Ca²⁺-dependent inactivation occurs at its maximal rate). We observed no acceleration of the Ca²⁺-dependent phase of current decay by 10 µmol/L or 50 µmol/L (-)gallopamil in _1L/ß_2a (_{Ca,50µmol/L,-10mV}=35±2 ms versus _{Ca,control,-10mV}=32±3 ms, _{Ca,50µmol/L,0mV}=45±6 ms versus _{Ca,control,0mV}=41±4 ms, P>0.05, Figure 4C, inset). Similar observations were made for _1L/ß_1a and _1L/ß₃ channels (data not shown).

(-)Gallopamil induced a concentration-dependent acceleration of the late phase of the I_Ca decay (_slow, Figures 4A and 4C) and accelerated the decay of I_Ba (Figures 4B and 4D). Interestingly, drug-induced and intrinsic voltage-dependent inactivation (Figures 4C, 4D, and 4D inset) displayed an almost identical voltage-dependence.

Permeant Ion Determines Recovery From the Drug-Induced Channel Conformation
To further elucidate the role of the charge carrying ion, we studied the recovery kinetics of I_Ba and I_Ca from inactivation in control and in the presence of (-)gallopamil (3 µmol/L to 50 µmol/L, Figure 5). In the absence of drug, I_Ba through _1L/ß_2a and _1L/ß₃ inactivated during a 3-second depolarisation to +10 mV between 15% to 25% (Figures 5A and 5B). Recovery at -80 mV was well-described by a single-exponential function with _Ba,ß2a=1.7±0.2 seconds (_1L/ß_2a, n=5) and _Ba,ß3=1.4±0.2 seconds (_1L/ß₃, n=6 Figures 5A and 5B, squares; Figure 5E and 5F, white bars). (-)Gallopamil increased the fraction of nonconducting channels at the end of the conditioning pulse in a concentration-dependent manner (Figure 5A through 5D). Recovery of drug-modified channels in 10 mmol/L Ba²⁺ was approximately monoexponential with corresponding time constants _{Ba,ß2a,(-)gallopamil}=2.8±0.3 seconds, n=14 and _{Ba,ß3,(-)gallopamil}=4.9±0.8 seconds, n=10 (Figures 5A, 5B, 5E, and 5F).

View larger version (27K): [in this window] [in a new window]   Figure 5. Effect of (-)gallopamil on Cav1.2 recovery from inactivation. Recovery of 1L/ß2a (A, C, and E) and 1L/ß3 (B, D, and F) in control (squares) and in the presence of 3 µmol/L (circles), 10 µmol/L (triangles) or 50 µmol/L (diamonds) (-)gallopamil estimated by a double-pulse protocol after a 3-second depolarization to the potential of maximal IBa (A and B) and ICa (C and D). Recovery kinetics in Ba2+ were fitted by a single-exponential function. Two-exponential fits were required to describe repriming with Ca2+ as charge carrier. The fast component represents recovery from Ca2+-dependent inactivation (1L/ß2a: Ca,ß2a=114±7 ms; 1L/ß3: Ca,ß3=104±13 ms) and the slow component represents recovery from voltage-dependent inactivation. (-)Gallopamil concentration-dependently diminished the amplitude of the fast Ca2+-dependent component. The insets show the relative amplitudes of the fast and slow (see Materials and Methods) recovery components in control and in the presence of 50 µmol/L (-)gallopamil. E and F, The time constants of IBa and ICa recovery from fast voltage-dependent inactivation in control (white bars) and from PAA-induced inactivation (black bars) of 1L/ß2a and 1L/ß3, respectively.

Because of Ca²⁺-dependent inactivation, a substantially larger fraction of channels inactivated during a 3-second test pulse to +20 mV in 10 mmol/L Ca²⁺ solution. The predominant fraction of channels recovered rapidly from Ca²⁺-dependent inactivation with respective time constants _Ca,ß2a=114±7 ms and _Ca,ß3=104±13 ms (n6) (Figure 5C and 5D). The second component in recovery occurred at a comparable rate as in 10 mmol/L Ba²⁺ solution and is, therefore, likely to reflect recovery from the voltage-dependent inactivation.

Two findings distinguished the recovery from channel inhibition by (-)gallopamil with Ca²⁺ as charge carrier. First, (-)gallopamil diminished the fraction of channels recovering from Ca²⁺-dependent inactivation in a concentration-dependent manner (Figures 5C and 5D, see insets). Second, recovery of drug-modified channels in 10 mmol/L Ca²⁺ solution occurred at a significantly slower rate (15±3 seconds versus 2.8±0.3 seconds [_1L/ß_2a], 16±3 seconds versus 4.9±0.8 seconds [_1L/ß₃], Figures 5E and 5F) than in Ba²⁺.

These observations suggest the possibility that Ca²⁺-dependent inactivation is modulated by (-)gallopamil. Indeed, one could hypothesize that the channels enter the Ca²⁺-dependent inactivated state at a similar rate as in control but recover slowly from a drug-modulated Ca²⁺-dependent inactivated state. To test this hypothesis, we analyzed the recovery from Ca²⁺-dependent inactivation of _1L/ß₃ after a short (100-ms) depolarizing conditioning pulse (Figure 6). In the absence of drug, a 100-ms pulse induced almost exclusively Ca²⁺-dependent inactivation (see rapid and complete recovery in Figure 6, squares). If (-)gallopamil were to change recovery from Ca²⁺-dependent inactivation, it would occur at a comparably slow rate as observed in Figure 5D (illustrated by the dashed line in Figure 6).

View larger version (14K): [in this window] [in a new window]   Figure 6. Recovery from Ca2+-dependent inactivation is not affected by (-)gallopamil. Recovery of 1L/ß3 from Ca2+-dependent inactivation after a 100-ms conditioning pulse estimated with a double-pulse protocol. In the absence of drug (squares), recovery is monoexponential (Ca,ß3=95±3 ms) and almost complete within 0.5 seconds. In the presence of 50 µmol/L (-)gallopamil (circles), recovery kinetics are well-fitted by single-exponential function with a similar time constant (Ca,ß3,50µmol/L=90±4 ms) and an offset (4±1%). The dashed line represents the recovery time course (=15 seconds) observed in Figure 5D.

This was, however, not the case (Figure 6, circles). Despite the high drug concentration the predominant fraction of the channels recovered at a fast rate that is typical for recovery from Ca²⁺-dependent inactivation in the absence of drug (_Ca,ß3=95±3 ms, _{Ca,ß3,50µmol/L}=90±4 ms, n=5). A similar observation was made in the presence of 100 µmol/L (-)gallopamil (data not shown). We, therefore, conclude that (-)gallopamil does not affect the recovery kinetics from Ca²⁺-dependent inactivation. The small fraction (4±1%) of slowly repriming channels apparently reflects recovery from PAA-induced inactivation accumulated during the 100 ms depolarizing pulse.

Slow Voltage-Dependent Inactivation and Ca_v1.2 Inhibition by (-)Gallopamil
To analyze a possible role of slow inactivation in Ca_v1.2 inhibition by (-)gallopamil, we extended the conditioning pulse to 30 seconds. The double-pulse protocol used for the estimation of recovery from slow inactivation (see Materials and Methods) incorporated a 10-second gap between the conditioning pulse and the first test pulse, enabling almost complete recovery of _1L/ß_2a and _1L/ß₃ channels from the fast voltage-dependent inactivation and inhibition by (-)gallopamil (_Ba,ß2a,drug=2.8±0.3 seconds, _Ba,ß3,drug=4.9±0.8 seconds, in Figures 5A and 5B). The remaining fraction of inactivated channels (between 20% and 30%) recovered from slow voltage-dependent inactivation. This recovery was monoexponential and occurred in control and in the presence of 10 µmol/L (-)gallopamil with identical kinetics (_{slow,ß2a,control}=25±3 seconds, _{slow,ß2a,10µmol/L}=27±4 seconds, P>0.05, Figure 7A; _{slow,ß3,control}=17±2 seconds, _{slow,ß3,10µmol/L}=19±4 seconds, P>0.05, Figure 7B).

View larger version (15K): [in this window] [in a new window]   Figure 7. Slow voltage-dependent inactivation is not affected by (-)gallopamil. Recovery from slow voltage-dependent inactivation was estimated after a 30-second conditioning pulse during a train of test pulses (30 ms) applied after the indicated recovery intervals in control (squares) and in the presence of 10 µmol/L (-)gallopamil (triangles) of (A) 1L/ß2a (slow,ß2a,control=25±3 seconds, slow,ß2a,10µmol/L=27±4 seconds, P>0.05) and (B) 1L/ß3 (slow,ß3,control=17±2 seconds, slow,ß3,10µmol/L=19±4 seconds, P>0.05).

We observed neither (-)gallopamil-induced changes in the kinetics of recovery from slow voltage-dependent inactivation, nor a drug-induced increase in the amount of slow-inactivated channels. Thus, PAA-induced inactivation and slow voltage-dependent inactivation are clearly distinguishable with respect to their recovery kinetics and, therefore, are likely to represent distinct processes.

	Discussion

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ß-Subunit Composition of Ca_v1.2 Affects Voltage-Dependent Inactivation and Apparent PAA Sensitivity
Our results demonstrate that the ß-subunit composition of Ca_v1.2 determines not only voltage-dependent inactivation kinetics but also their sensitivity for the PAA (-)gallopamil (Figures 1 and 2). First evidence for ß-subunit–dependent PAA sensitivity was reported by Lacinova et al,²⁵ who observed a higher sensitivity of Ca_v1.2 for (-)gallopamil and verapamil when the ₁1.2-subunit was coexpressed together with the ß₃-subunit compared with cells transfected solely with ₁1.2. We have later demonstrated that the PAA sensitivity of a mutant ₁2.1 construct is determined by ß-subunit–dependent inactivation properties (_1A-PAA).¹³ The results of the present study extend those previous observations to Ca_v1.2 channels. Our data show that Ca_v1.2 constructs formed by ß_1a- and ß₃-subunits (promoting fast voltage-dependent inactivation) are more efficiently inhibited by (-)gallopamil than _1L/ß_2a channels (Figure 2).

Selective Interaction of (-)Gallopamil With Fast Voltage-Dependent Inactivation
Our data suggest a selective modulation of the fast voltage-dependent inactivation mechanism by (-)gallopamil. (1) In line with earlier studies,^26,27 we found no drug-induced acceleration of the Ca²⁺-dependent component of I_Ca decay (Figures 4A and 4C). (2) The kinetics of recovery from Ca²⁺-dependent inactivation were unaffected even by high drug concentrations (50 to 100 µmol/L, Figure 6). (3) (-)Gallopamil concentration dependently increased a slow component in channel recovery (likely to represent recovery from the fast voltage-dependent inactivation) with Ca²⁺ and Ba²⁺ as charge carriers (Figure 5). (4) In accordance with early work of Lee and Tsien⁴ and other groups,^7,11,12,28 we observed a pronounced drug-induced acceleration of the voltage-dependent component of current decay with Ba²⁺ and Ca²⁺ as charge carriers (Figures 4A through 4D). (5) The impaired fast voltage-dependent inactivation produced by the _1L-ß_2a interaction correlates with the decrease in the apparent (-)gallopamil sensitivity (Figure 2). (6) PAA-induced acceleration of the current decay and intrinsic fast inactivation displayed a similar voltage-dependence (Figures 4C and 4D). (7) Finally, neither the amount of slow inactivated channels nor the kinetics of recovery from slow inactivation were affected by (-)gallopamil (Figure 7).

Together, these data show that fast voltage-dependent inactivation is of key importance for the drug-channel interaction, whereas Ca²⁺-dependent and slow inactivation are not modified by (-)gallopamil.

Role of the Permeant Ion in PAA Sensitivity
If Ca²⁺-dependent inactivation is virtually unaffected by (-)gallopamil, how can the enhanced use-dependent inhibition of Ca_v1.2 constructs by (-)gallopamil with Ca²⁺ as charge carrier (Figure 2) be explained? Our data demonstrate that I_Ca recover from inhibition by (-)gallopamil at a rate about 3 to 5 times slower than I_Ba (Figures 5E and 5F), suggesting that a decelerated recovery from PAA inhibition is the mechanism underlying the enhanced apparent PAA sensitivity in Ca²⁺ versus Ba²⁺ (Figure 2, see also Lee and Tsien⁴ and Nawrath and Wegener.²⁷

One possible interpretation of this finding is that an interaction of Ca²⁺ with a putative drug binding site increases the affinity of the receptor for the drug (as originally postulated by Glossman and Ferry²⁹) resulting in a higher stability of the drug-channel complex. Alternatively, the interaction of the channel with a divalent cation could affect the ability of the drug to induce inactivation (efficacy, see Colquhoun³⁰ for review). Our experimental data do not allow these possibilities to be to distinguished between. Moreover, it is currently not clear if the increase in apparent drug sensitivity with Ca²⁺ is accomplished by the ion interaction with pore determinants (eg, Glu¹¹¹⁸ and Glu¹⁴¹⁹; see Hockerman et al³¹) or, alternatively, with a site that is located in the region of the C-terminus.^22,24,32,33

Simulation of PAA-Induced Effects on Channel Inactivation
The molecular mechanism of Ca_v1.2 inhibition by PAAs is conventionally explained by high-affinity drug binding to open or inactivated channels.^{2–4,7,8,26,27,34}

A high-affinity drug binding to the inactivated channel state would result in a substantial shift of the inactivation curve to more negative potentials. However, if the inactivation curve is measured under conditions close to steady-state the observed shift is hardly significant³⁵ (see also Aczel et al¹⁰ and Degtiar et al³⁶ for an overview of the steady-state problem).

The open-channel block hypothesis does not account for the recently observed correlation between the rate of intrinsic channel inactivation and the apparent drug sensitivity.^11,12,28 Most strikingly, the considerably larger fraction of open channels in Ca_v1.2 composed of ß_2a- (compared with ß_1a- and ß₃-) subunits did not result in a higher PAA sensitivity. Instead, a marked reduction of use-dependent channel inhibition was observed (Figure 2, see also Motoike et al,¹¹ Hering et al,¹² and Figure 1D in Sokolov et al¹³). These data suggest an important role of the inactivation machinery in the drug-channel interaction.

An alternative concept to explain the action of (-)gallopamil is to assume that the drug promotes intrinsic fast voltage-dependent inactivation. In the model illustrated in Figure 8A, the drug binding/unbinding to the channel is assumed to occur in a state-independent manner. Equilibrating a cell membrane in drug produces a certain steady-state fraction of drug-modified channels. Therefore, at each particular drug concentration two populations of channels (modified and non-modified channels) coexist in a dynamic equilibrium.

View larger version (24K): [in this window] [in a new window]   Figure 8. Simulation of the use-dependent inhibition of Cav1.2 by (-)gallopamil. A, The kinetic model of channel inactivation incorporates Ca2+-dependent (Ca-I), fast voltage-dependent (Fast-I), and slow inactivation (Slow-I). Only the microscopic rates and are affected by the drug. B, Simulation of use-dependent inhibition of 1L/ß3 and 1L/ß2a by 10 µmol/L (-)gallopamil with Ba2+ or Ca2+ as charge carrier. Experimental traces on the left are shown for comparison. The effect of the ß2a-subunit is simulated as a destabilization of the fast inactivated state by decreasing and increasing at depolarized potential. C, Simulation of 1L/ß3 recovery kinetics (dashed line) in control (squares) and in the presence of 3 µmol/L (circles), 10 µmol/L (triangles), and 50 µmol/L (diamonds) (-)gallopamil with Ba2+ or Ca2+ as charge carrier. See online data supplement for the full set of fit parameters.

The basic assumption of the model is that (-)gallopamil stabilizes the fast voltage-dependent inactivation. Consequently, the drug-induced acceleration of the current decay is simulated by increasing the onset of fast inactivation (rate ) (_1L/ß₃: _Ba,control=0.13 s^-1 versus _Ba,drug=0.85 s^-1) and the slower recovery by a deceleration of the recovery (rate ) in the fraction of drug-modified channels (_1L/ß₃: _Ba,control=1.25 s^-1 versus _{Ba drug}=0.2 s^-1, see online data supplement for full set of fit parameters, available at http://www.circresaha.org).

To account for Ca²⁺-dependent inactivation, we have extended our previous inactivation model³⁷ by introducing a Ca²⁺-dependent inactivated state. Although (-)gallopamil does not affect the microscopic rates of Ca²⁺-dependent inactivation (rate ) and recovery (rate ß) (see Figure 4A and 4C inset and Figure 6), the model nicely reproduces the higher use-dependent channel block in Ca²⁺ compared with Ba²⁺ (Figure 8B). The I_Ba recovery kinetics in the presence of 3, 10, and 50 µmol/L (-)gallopamil (corresponding to increasing fractions of drug-modified channels) are simulated in Figure 8C, left panel (data from Figure 5B). As shown in the right panel of Figure 8C, the slower recovery of drug-modified channels in Ca²⁺ (data from Figure 5D) can be reproduced by a further reduction of the microscopic rate at hyperpolarized voltages (_1L/ß₃: _Ba,drug=0.2 s^-1 versus _Ca,drug=0.06 s^-1).

One of the intriguing results of our simulation is that the difference in apparent PAA sensitivity of _1Lß_2a and _1Lß₃ channels does not require to assume that the ß-subunit interaction affects the affinity of the channel for (-)gallopamil (ie, drug binding step). Instead, the lower apparent sensitivity of _1Lß_2a channels may be equally well-explained by an interplay of the kinetic rates and . This finding is illustrated in Figure 8B, where the ß_2a-subunit effect on channel gating was simulated as a destabilization of the fast inactivated state (see also previous observations of Berjukow et al³⁸ in Ca_v1.2). Decreasing and increasing reproduced not only the slower inactivation kinetics of _1L/ß_2a in control but simultaneously resulted in less use-dependent channel inhibition in the presence of drug (see simulated _1L/ß_2a traces in Figure 8B).

Taken together, we present experimental evidence that different ß-subunit compositions of Ca_v1.2 affect not only the channel inactivation kinetics but also the PAA sensitivity. This finding may be of high pharmacological relevance because ₁1.2 appear to be associated with distinct ß-subunits in different tissues.³⁹ It will be interesting to analyze if differences in drug-sensitivity of Ca_v1.2 (eg, in heart and smooth muscle preparations) are related to a different ß-subunit composition. The precise ß-subunit composition of Ca_v1.2 under physiological conditions is, however, mostly unknown. Immunoprecipitation studies suggest that only about 50% of ₁1.2 in porcine heart but almost all in the uterus are associated with the ß₂-isoform.³⁹ Recent functional studies of Wei et al⁴⁰ demonstrated, however, that other isoforms than ß_2a are functionally dominant in native rat heart. In smooth muscle (trachea, aorta) immunoprecipitation studies suggest an interaction with unknown ß-subunits. Thus, more detailed knowledge about the ß-subunit composition of Ca_v1.2 is required before the data presented here can be correlated with a pharmacological data obtained from native tissues.

Irrespective of their ß-subunit composition we observed a higher PAA sensitivity with Ca²⁺ than with Ba²⁺ as charge carrier reflecting a Ca²⁺-mediated stabilization of a PAA-induced conformation. We show here that only the voltage-dependent phase of I_Ca decay (as well as the voltage-dependent decay of Ba²⁺ currents) is accelerated by (-)gallopamil. This finding explains contradictions in a number of previous studies where some authors working in Ba²⁺ solution reported a pronounced PAA-induced acceleration of current kinetics,^4,7,11,12,28 whereas the others working with short pulses in Ca²⁺ observed no acceleration.^26,27 Our simulation study demonstrated that the molecular mechanism of (-)gallopamil action is well explained by a drug-induced stabilization of the fast voltage-dependent inactivated state (Figure 8).

	Acknowledgments

 
This work was supported by FWF-grants P12649-MED and P12828-MED, a Grant from the Else Kröner-Fresenius-Stiftung, and a grant of the Austrian National Bank (to S.H.). The authors thank Prof H. Glossmann for continuous support and Dr S. Berjukow for helpful suggestions and comments on the manuscript.

Received January 22, 2001; revision received May 31, 2001; accepted September 10, 2001.

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