Introduction

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In cardiovascular cells, DHPs block L-type Ca²⁺ channels more potently at depolarized membrane potentials (Bean, 1984; Hess et al., 1984; Sanguinetti and Kass, 1984; Schwartz et al., 1984). This observation has been interpreted as evidence that DHPs bind with high affinity to the inactivated state of the channels (Bean, 1984; Sanguinetti and Kass, 1984). If so, DHPs would interact with Ca²⁺ channels in a manner analogous to the interaction between local anesthetics and Na⁺ channels (Hille, 1977). DHPs function more potently on SM than on cardiac muscle or neurons (Triggle, 1991); indeed, such specificity has been suggested to reflect voltage-dependent inhibition because, in general, the resting membrane potential of SM cells is more positive than that of myocardial cells (Nelson et al., 1988; Triggle, 1991).

The cloning and functional expression of Ca²⁺ channels have made it possible to study isoform-specific pharmacological properties in well controlled systems. L-type Ca²⁺ channels are composed of three to four subunits; the pore-forming 1 is the primary subunit and the rest (2/, , and/or ) are auxiliary subunits (Perez-Reyes and Schneider, 1995; Catterall, 1996). Cardiac and SM channels share  subunit isoforms (2a) and seem not to express the  subunit (Perez-Reyes and Schneider, 1995). The 1 subunit is the target of many drugs, including DHPs (Vaghy et al., 1987; Triggle et al., 1989). The 1C-a (Mikami et al., 1989) and the 1C-b (Biel et al., 1990) isoforms are alternatively spliced from the same 1C gene, with 95% similarity. The 5% differences are located at four sites, which are in the amino terminus, the transmembrane segments IS6 and IVS3, and an insert in the linker connecting domains I and II for 1C-b. Studies of cloned Ca²⁺ channels expressed in mammalian cells indicate that 1C subunits alone are sufficient to produce the tissue-specific DHP sensitivity difference (Welling et al., 1993, 1997). Specifically, with 1C-a or 1C-b alone in Chinese hamster ovarian cells and 30 mM Ba²⁺ as the charge carrier, nisoldipine blocked both isoforms of the channel more potently at 40 mV holding potential than at 80 mV, with a higher affinity for the 1C-b channels. Recently, Welling and colleagues (1997) reproduced these findings in the HEK 293 expression system, using 1C subunits truncated at the carboxyl-terminal. They further identified the IS6 segment of the 1C subunit as the agent responsible for the tissue specificity of nisoldipine inhibition.

Both voltage-dependent and tissue-specific nisoldipine inhibition of Ca²⁺ currents in native channels and Ba²⁺ currents in expressed channels has been reported. Nevertheless, there is no direct evidence linking or separating the tissue specificity from possible isoform-specific differences in gating. To investigate the links between isoform-specific gating and drug block, we expressed 1C-a or 1C-b + 2a + 2/ channels in HEK 293 cells, and used 2 mM Ca²⁺ as the charge carrier to achieve more physiologically relevant conditions. This system is thus close to the in vivo topology of the channels, yet contrasting the two 1C splice variants is much easier than in native cells.

We are interested in elucidating (1) whether voltage-dependent and isoform-specific DHP inhibition can be reproduced in our expression system and (2), more importantly, whether the isoform specificity is due to differences in gating.

	Materials and Methods

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Functional expression of cardiac and smooth muscle Ca²⁺ channel 1C subunits. The transient transfection of L-type Ca²⁺ channels in HEK 293 cells was performed as described previously (Hu et al., 1997). Briefly, HEK 293 cells were maintained in Dulbecco's modified Eagle's medium with glucose and L-glutamine, supplemented with 10% fetal calf serum (GIBCO-BRL, Gaithersburg, MD) and 1% penicillin and streptomycin (GIBCO-BRL). Cells were plated on 35-mm Petri dishes at a density of 0.2 million cells/dish 1 day before transfection, and maintained in a 37° incubator. Cells were then transfected by calcium phosphate precipitation (Graham and van der Eb, 1973; Calcium Phosphate Transfection System, GIBCO-BRL) with 2-3 µg/dish plasmid DNA encoding Ca²⁺ channel subunits (see below), 0.5 µg/dish simian virus 40 T-antigen, and 0.2 µg/dish mitochondrially targeted green fluorescent protein (Marshall et al., 1995). The calcium phosphate-DNA mixture was left on cells for 5-6 hr before being washed with phosphate-buffered saline and the addition of fresh media. The admixture of green fluorescent protein cDNA enabled us to identify transfected cells visually by fluorescent excitation (Marshall et al., 1995).

Electrophysiology and data processing. Electrophysiological recordings were made 18-72 hr after transfection. Membrane current was recorded using the whole-cell patch configuration (Hamill et al., 1981), with bath solution containing 2 mM CaCl₂, 147 mM CsCl, and 10 mM HEPES (titrated to pH 7.4 with CsOH). Pipettes were pulled from borosilicate glass and fire-polished to resistance of 0.5-2 M when filled with pipette solution containing 108 mM CsCl, 4.5 mM MgATP, 9 mM EGTA, and 9 mM HEPES (titrated to pH 7.4 with CsOH). In the whole-cell configuration, the series resistance was typically 2-5 M. In most of the experiments the series resistance was not compensated; this would have introduced a maximal voltage error of < 2.5 mV as the peak current magnitude was generally < 500 pA.

Statistics. Pooled data are presented as mean ± standard error. Statistical comparison was evaluated by the two-tailed paired or unpaired Student's t test, or ANCOVA, where appropriate, with p < 0.05 considered significant.

	Results

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We first confirmed and characterized the voltage dependence of nisoldipine inhibition on Ca²⁺ channels in our expression system. Fig. 1A shows the effect of 10 nM nisoldipine on I_Ca through 1C-a + 2a + 2/ channels, which is the cardiac isoform. In Fig. 1, the top shows representative currents recorded at the times indicated at the bottom. The voltage protocol used is illustrated on top of record a. Fig 1A, bottom, plots the time course of peak I_Ca. The cell was first held at 100 mV, a V_h at which the channels were fully reprimed (trace a). Then the V_h was changed to 65 mV (); slow inactivation was observed, stabilizing in 3-4 min, with a relative remaining current of 49% (trace b). Introduction of 10 nM nisoldipine potently blocked the current at V_h = 65 mV (7% remaining, trace c), yet when the cell was repolarized to V_h = 100 mV, an unblocking effect was observed, and I_Ca recovered to 48% of the original amplitude, despite the fact that 10 nM nisoldipine was still present (trace d). Note that polyethylene glycol 400 itself, as the solvent for nisoldipine, has no effect on I_Ca even at concentrations 1000 times higher than those used in the present study (Kass and Scheuer, 1982).

View larger version (17K): [in this window] [in a new window]   Fig. 1.   A, Effects of nisoldipine and Vh on ICa from the cardiac channel 1C-a + 2a + 2/ expressed in HEK 293 cells. Top, Representative currents from Vh = 100 mV (a, d) or 65 mV (b, c) at steady state, in the absence (a, b) or presence (c, d) of 10 nM nisoldipine, recorded at the times a, b, c, and d indicated in B. The voltage protocol is shown on top of trace a. The records shown were smoothed using a two-adjacent-point averaging method. Bottom, Time course of ICa. The cell was first held at 100 mV () and then 65 mV () in normal saline. After steady state inactivation was reached at 65 mV, 10 nM nisoldipine solution was applied and the current was dramatically suppressed. Still in the presence of nisoldipine, changing Vh back to 100 mV largely unblocked the current. B, Effects of nisoldipine and Vh on ICa from the SM channel 1C-b + 2a + 2/ expressed in the HEK 293 cells. Top, Representative currents from Vh = 100 mV (a, d) or 65 mV (b, c) at steady state, in the absence (a, b) or presence (c, d) of 10 nM nisoldipine, recorded at the times a, b, c, and d indicated. The voltage protocol is shown above trace a. Unlike those in Fig. 1A, records plotted here are not smoothed. Bottom, Time course of ICa. The cell was first held at 100 mV () and then 65 mV () in normal saline. After steady state inactivation was reached at 65 mV, 10 nM nisoldipine solution was applied and the current was almost completely inhibited. Still, in the presence of nisoldipine, changing Vh back to 100 mV partly unblocked the current.

Despite the differences in the absolute V_h values chosen here compared with those in previous studies (Welling et al. 1993, 1997), these findings are entirely consistent with earlier studies after correction for surface charge effects on the cell membrane potential. Recall that previous studies, which used 80 and 40 mV holding potentials, were performed with 30 mM Ba²⁺ as the charge carrier, which would be expected to cause an ~20 mV depolarizing voltage shift (Kass and Krafte, 1987; McDonald et al., 1994).

Using the same protocol, we examined the effect of nisoldipine on I_Ca through the SM channel (1C-b + 2a + 2/). Fig. 1B shows a representative experiment. Three differences were observed when these results were compared with those of the cardiac channels. First, the SM channel inactivated less at V_h = 65 mV; second, 10 nM nisoldipine blocked the SM channels more completely than the cardiac channels; third, after the cell was repolarized to V_h = 100 mV, the unblocking effect was less prominent.

Fig. 2 summarizes the effects of nisoldipine inhibition on the two channel isoforms. For both, nisoldipine blocked more potently at 65 mV, when channels were partly inactivated, than at 100 mV when the channels were fully reprimed, in line with the idea of inactivated-state inhibition. Among the three differences between the two channel isoforms, the first (steady state inactivation at 65 mV) was not statistically significant, yet the probability of null hypothesis p = 0.056 indicates a strong trend that the cardiac channel inactivated more than the SM one at V_h = 65 mV. The second difference (inhibition at V_h = 65 mV) and the third (inhibition at V_h = 100 mV) were unambiguously significant, and they agree with previously published results (Welling et al., 1993, 1997). The first difference, that in drug-free steady state inactivation, would thus not help to explain the second difference if the nisoldipine inhibition depended mainly on the inactivated state as suggested by studies in native cells (Bean, 1984; Sanguinetti and Kass, 1984): when channels are more inactivated, block by nisoldipine should be more potent, not less as observed here.

View larger version (21K): [in this window] [in a new window]   Fig. 2.   Pooled data summarizing steady state effects of nisoldipine and Vh on cardiac and SM channels. ICa elicited from Vh = 100 mV to 0 mV in normal saline was used as the reference current (100%), and Irelative was defined as the ratio of the remaining current to the reference current. Three pairs of bars represented relative currents at Vh = 65 mV, in the presence of 10 nM nisoldipine while Vh was still 65 mV, and when Vh was changed back to 100 mV while 10 nM nisoldipine was still present. *, statistically significant. Note that the SM channel was moderately less inactivated at 65 mV than the cardiac one, with a marginal p value of 0.056.

To extend these observations, we quantified the voltage dependence of inactivation of both types of channels under drug-free conditions. The results shown in Fig. 1 indicate that channels inactivate with two distinct time courses during changes of V_h from 100 to 65 mV. A fast component of inactivation, which appears within seconds, is followed by a slower component, which is complete only over ~3 min. We first measured the fast inactivation curves of the channels, using 1-sec prepulses from V_h = 80 mV; the results are shown in Fig. 3A, with the voltage protocol illustrated as an inset. For each channel type, 10 complete individual inactivation curves were obtained. The pooled data of inactivation were normalized to enable the Boltzmann fits of the curves to reach unity, with an equation of I_relative = A + (1A)/[1 + exp(V_pV_1/2)/k] where I_relative is the relative remaining current, A is the fraction of the incomplete inactivation, V_p is the prepulse membrane potential, V_1/2 is the potential at which one-half of the channels are inactivated, and k is the slope. The fitting of the mean inactivation for the cardiac channels gave V_1/2 = 35 mV, k = 17 mV, and A = 0.32, and for the SM channels, V_1/2 = 30 mV, k = 9 mV, and A = 0.29. There is a trend that, at voltages more negative than 30 mV, the cardiac channels are more inactivated, but there were no significant differences in either V_1/2 or k in the two groups.

View larger version (21K): [in this window] [in a new window]   Fig. 3.   Inactivation of the two isoforms of the channels. A, Fast inactivation of the cardiac (, n = 10) and SM (, n = 10) channels. The smooth curves represent Boltzmann fits. Paired t test of derived Boltzmann parameters (V1/2 and k) from the original inactivation curves showed no difference between the two channel isoforms. Inset, double-pulse voltage protocol used in these experiments, which is composed of a 0-mV test pulse of 15 msec, a 80-mV reactivation pulse of 200 msec, a 1-sec inactivation prepulse to potential Vp, and a second 0-mV test pulse of 25 msec. B, Slow inactivation of the cardiac () and SM () channels (see text). Pulses to Vp here lasted 3 min. Data were recorded from 12 cells expressing the cardiac channel and 14 cells expressing the SM channel. Points, averages of data from three to six cells. ANCOVA showed no significant differences between the two groups of data.

Because Fig. 1 shows that it takes several minutes to reach steady state inactivation, we considered not only fast inactivation but also the less well-characterized slow inactivation process. We therefore studied inactivation of the channels with 3 min prepulses. The corresponding recovery was slower when compared with that needed for fast inactivation, and we allowed 5 min for recovery at 80 mV between pulses. A brief test pulse (10 msec) to 0 mV was applied every 20 sec to monitor the recovery at 80 mV. The two steady state currents elicited from 80 mV on either side of each inactivation prepulse were averaged to obtain the reference current.

Fig. 3B shows the results of 3-min prepulse inactivation. Compared with Fig. 3A, the prolonged inactivation shifted the curves dramatically to the left, and the saturating inactivation reached 100% instead of 60-70% as in the fast inactivation. Data were collected from 12 cells expressing cardiac channels and 14 cells expressing SM channels; each point represents data from 3 to 6 cells. Boltzmann fitting of the mean values using I_relative = [1 + exp(V_p  V_1/2)/k]^-1 gave V_1/2 = 57 mV and k = 9 mV for the cardiac channels, and V_1/2 = 49 mV and k = 10 mV for the SM channels. ANCOVA, however, did not show a significant difference between the two original groups of data. There was a trend, however, for the cardiac curve to be shifted to the left compared with that of the SM channel; once again, those differences were in the wrong direction to help rationalize the voltage-dependent block by nisoldipine.

Given these observations, we had to conclude that the sensitivity difference to nisoldipine block of the two isoforms lies in some mechanism other than the inactivation gating of the channels. One possibility is the activation gating of the channels, which can directly or indirectly influence voltage-dependent drug actions (Kuo and Bean, 1994; Balser et al., 1996), might differ in the two isoforms. We thus studied the current-voltage relations of the channels and deduced the activation curves from them (Fig. 4, A and B). The conductance-voltage curves, fit using G = [1 + exp(V_1/2V)/k]^-1, yielded parameters of V_1/2 = 16 mV and k = 8 mV for the cardiac channel, and V_1/2 = 17 mV and k = 7 mV for the SM one. The two channel isoforms are thus virtually identical in their activation properties.

View larger version (20K): [in this window] [in a new window]   Fig. 4.   Current-voltage relation and activation of the channels. A, Normalized current-voltage (I-V) relations of the cardiac () and the SM channel (). There were no statistical differences between the curves. B, Normalized conductance-voltage (G-V) relations of the cardiac () and the SM channel (). The conductance was deduced from the data in A using G = I/(VVrev), where I is the current, G is the conductance, V is the membrane potential, and Vrev is the reversal potential. Smooth curves are Boltzmann fits. There are no significant differences between the two groups of data.

	Discussion

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Our results demonstrate that isoform-specific sensitivity to DHP block is not caused by inactivation gating differences between the two channels. If anything, the cardiac (1C-a) channels tended to inactivate at more negative potentials than the SM (1C-b) channels, which would predict that nisoldipine would block the cardiac channel more potently. This prediction is the opposite of what we observed and of what has been described previously. We therefore have to propose that factors other than inactivation gating underlie the isoform-specific nisoldipine inhibition of L-type Ca²⁺ channels.

Our results indicate that some of the inactivation characteristics of the expressed channels are different from those reported previously in native cells. Fig. 3 shows that, for either type of the expressed channels, the inactivation was about 30% incomplete even after one second prepulses to 0 mV, and the slope of the cardiac channel inactivation was 17 mV. This is quite different from observations made in native cells, in which channels are generally completely inactivated under similar conditions, and the slope is ~7 mV (McDonald et al. 1994). There are reports, however, that when briefer prepulses (100-200 msec) were applied, 20% of channel activity remained (Lee et al., 1985). Interestingly, a similar amount of remaining channel activity was observed in a Xenopus laevis oocyte expression system with prepulses as long as 5 sec (Parent et al., 1995). Although there seem to be genuine (and unexplained) differences between native currents and those in heterologous expression systems, such differences do not affect our conclusions, which rely on internal comparisons within the HEK 293 expression system.

Welling et al. (1997) have identified transmembrane segment IS6 of the channels as responsible for the nisoldipine sensitivity differences by expressing carboxy-terminal truncated 1C-a or 1C-b subunits alone in HEK 293 cells. The 1C-a IS6 and 1C-b IS6 segments are alternatively spliced and selectively expressed in cardiac or SM cells. Although it has been suggested that this segment is involved in voltage-dependent inactivation (Zhang et al., 1994; Parent et al., 1995), our results unambiguously divorce any possible impact of inactivation from isoform-specific inhibition by DHPs. Our results also suggest that activation gating differences are not responsible for the isoform-specific inhibition. Thus, the isoform-specific inhibition by DHPs is not modulated by channel state or gating, although in general, the DHP inhibition of the channels is still heavily modulated by the membrane potential.

By exclusion, we conclude that the isoform-specific inhibition reflects an intrinsic DHP binding affinity difference between the two channel isoforms. The precise structural features of the DHP binding site remain to be determined. DHPs act on Ca²⁺ channels preferentially from the extracellular side of the cell (Kass and Arena, 1989). Photoaffinity and mutational analysis have suggested that the DHP binding domain is possibly composed of transmembrane segments IS6, IIIS6, IVS6, and the linker of IIIS5 and IIIS6 (Nakayama et al., 1991; Striessnig et al., 1991; Kalasz et al., 1993; Schuster et al., 1996), but not a cytosolic peptide directly after IVS6 as previously reported (Regulla et al., 1991). It is not yet clear whether transmembrane segment IS6 or other parts of the channel are the critical features of the DHP-binding domain that confer voltage-dependent inhibition.