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0026-895X/97/050872-10$3.00/0
Copyright © by The American Society for Pharmacology and Experimental Therapeutics
All rights of reproduction in any form reserved.
MOLECULAR PHARMACOLOGY 51:872-881 (1997).

Inhibition of Recombinant Ca2+ Channels by Benzothiazepines and Phenylalkylamines: Class-Specific Pharmacology and Underlying Molecular Determinants

Dongming Cai, Jennifer G. Mulle, and David T. Yue

Program in Molecular and Cellular System Physiology, Department of Biomedical Engineering, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205

    Summary
Summary
Introduction
Materials & Methods
Results
Discussion
References

To understand the molecular basis of state-dependent pharmacological blockade of voltage-gated Ca2+ channels, we systematically characterized phenylalkylamine and benzothiazepine inhibition of three molecular classes of Ca2+ channels (alpha 1C, alpha 1A, and alpha 1E) expressed from cDNA clones transfected into HEK 293 cells. State-dependent blockade figures importantly in the therapeutically desirable property of use-dependent drug action. Verapamil (a phenylalkylamine) and diltiazem (a benzothiazepine) were imperfectly selective, so differences in the state dependence of inhibition could be compared among the various channels. We found only quantitative differences in pharmacological profile of verapamil: half-maximal inhibitory concentrations spanned a 2-fold range (70 µM for alpha 1A, 100 µM for alpha 1E, and 110 µM for alpha 1C), and inhibition was state dependent in all channels. In contrast, diltiazem produced only state-dependent block of alpha 1C channels; alpha 1A and alpha 1E channels demonstrated state-independent block despite similar half-maximal inhibitory concentrations (60 µM for alpha 1C, 220 µM for alpha 1E, and 270 µM for alpha 1A). To explore the molecular basis for the sharp distinction in state-dependent inhibition by diltiazem, we constructed chimeric channels from alpha 1C and alpha 1A and localized the structural determinants for state dependence to repeats III and IV of alpha 1C, which have been found to contain the structures required for benzothiazepine binding. We then constructed a mutant alpha 1C construct by changing three amino acids in IVS6 (Y1490I, A1494S, I1497M) that have been implicated as key coordinating sites for avid benzothiazepine binding. Although these mutations increased the half-maximal inhibitory concentration of diltiazem inhibition by ~10-fold, the state-dependent nature of inhibition was spared. This result points to the existence of physically distinct elements controlling drug binding and access to the binding site, thereby favoring a "guarded-receptor" rather than a "modulated-receptor" mechanism of drug inhibition.

    Introduction
Summary
Introduction
Materials & Methods
Results
Discussion
References

Pharmaceutical agents that modulate voltage-gated Ca2+ channels have found widespread application as basic research tools and therapeutic drugs. From the biophysical perspective, L-type Ca2+ channel modulation by the most established generation of organic Ca2+ channel blockers (dihydropyridines, phenylalkylamines, and benzothiazepines) provides rich experimental models of allosteric modification of gating and/or physical blockade of the conduction pathway. In physiological experiments, these organic compounds, together with neuroactive peptides that block specific molecular classes of Ca2+ channels (1), have been used to delineate the contributions of different channel types to specific biological functions. In the clinical setting, members of each of the three classes of organic compounds act on L-type channels to treat disorders as varied as angina, hypertension, and cardiac arrhythmia (2-4). Moreover, recombinant neuropeptides that inhibit neuronal N-type channels hold promise for the treatment of chronic intractable pain (5). Finally, mutations in neuronal P/Q-type channels have been linked to a inherited forms of migraine and ataxia (6), raising the possibility that as-yet-undiscovered compounds that select for P/Q channels may provide novel therapies for the more general forms of these neurological disorders. For all these reasons, there is enormous interest in understanding the molecular basis of Ca2+ channel inhibition and discovering new compounds with high specificity for selected molecular classes of Ca2+ channels.

The cloning and expression of several classes of Ca2+ channels provide powerful tools for addressing just these issues. First, mutant and chimeric Ca2+ channel analysis on the main alpha 1 subunits have already revealed several key amino acid residues that can account for much of the class-specific difference in binding affinity of dihydropyridines (7-9), phenylalkylamines (9-11), and benzothiazepines (12, 13). Second, the ability to express a homogeneous population of a selected type of channel, in the virtual absence of contaminating currents, provides an ideal opportunity to assess the relative effect of an agent on a particular class of Ca2+ channel. The coexistence of multiple channel types in neurons and other native cells may complicate quantitative attribution of drug inhibition to particular channel types.

Despite the rapid advances and molecular approaches outlined above, there is still little understanding of the molecular mechanism of Ca2+ channel inhibition. All recombinant channel studies to date have focused mainly on simple differences in the potency of inhibition of different recombinant Ca2+ channels by various organic blockers. No systematic comparison has been made of the characteristic phenotype of block. In particular, there is little information on the relative state dependence of block, a feature that is critical to the therapeutically useful property of "use dependence" (16, 17). Use-dependent blockers preferentially inhibit channels during unusually high electrical activity, as found in a cardiac arrhythmia or seizure focus. Differences in state-dependent block among the various recombinant channels could point the way to the structural determinants of use dependence. These molecular elements may be distinct from the structures that specify simple binding affinity according to the guarded-receptor hypothesis (18).

Here, we report the systematic characterization of phenylalkylamine and benzothiazepine inhibition of three molecular classes of Ca2+ channels expressed in HEK 293 cells from cDNA clones encoding alpha 1A, alpha 1E, and alpha 1C subunits. The first two alpha 1 clones correspond to neuronal P/Q-type and R-type channels (19), and the latter clone corresponds to cardiac L-type channels. We chose to study these two classes of organic compounds because they proved to be imperfectly selective, so differences in the state dependence of block could be compared among the various classes of Ca2+ channels. Dihydropyridines were almost perfectly selective for L-type channels (7; but see 14, 15), so no such comparison can be made.

    Materials and Methods
Summary
Introduction
Materials & Methods
Results
Discussion
References

Expression of recombinant voltage-gated Ca2+ channels. cDNAs encoding calcium channel alpha 1, beta 2a (20), and alpha 2 (21) subunits were subcloned into mammalian expression plasmids. alpha 1C (22), alpha 1E (23), and beta 2a were subcloned into cytomegalovirus-promotor expression plasmid pGW1H (British Biotechnologies, Oxford, UK); alpha 1A (24, 25), as well as chimeric and/or mutant alpha 1 subunits (alpha 1CCAA, alpha 1AACC, and alpha 1C-ISM, as described below), was subcloned into CMV-promotor expression plasmid pcDNA3 (InVitrogen Corporation, San Diego); and alpha 2 was subcloned in the constitutively active, metallothionein-promotor expression plasmid pZEM229R (Zymogenetics, Seattle, WA). Low-passage-number (< 20) HEK 293 cells, obtained from Dr. Jeremy Nathans (26), were transiently transfected with plasmids containing alpha 1 and beta 2a subunits (10 µg each/10-cm plate) using a calcium-phosphate precipitation procedure (27). With chimeric channels, the alpha 2 subunit and pAdVAntage vector (Promega, Madison, WI) were sometimes cotransfected (10 and 5 µg/10-cm plate, respectively) to enhance expression, as noted. Cotransfection of the alpha 2 subunit had no detectable effect on the pharmacological profile of diltiazem (not shown). pAdVAntage encodes the transcription-enhancing genes VAI and VAII from the adenovirus genome (28). Recombinant currents were observed in > 30% of transfected cells for the majority of constructs; transfection with a beta -galactosidase reporter gene resulted in a > 50% overall expression rate.

Mock-transfected cells were cotransfected with beta 2a and alpha 2 subunits. No high-voltage-activated channels were detected (n = 25 cells in three rounds of transfection). In mock-transfected cells, we occasionally (~10-20% of cells) observed endogenous, low-voltage-activated Ca2+ currents of small amplitude. Although endogenous currents of such small amplitude would contribute negligibly to most of our results, such cells were nevertheless rejected. Therefore, all data reflect the expression of recombinant Ca2+ channels.

Construction of mutant and chimeric Ca2+ channel alpha 1 subunits. Chimeric Ca2+ channels were generated in which repeats I and II were interchanged between alpha 1C and alpha 1A, yielding alpha 1CCAA and alpha 1AACC (see Fig. 8, top). To construct alpha 1CCAA, we amplified a region spanning repeats III and IV of alpha 1A (nucleotides 2024-6639, with nucleotide 1 at the start codon, here and throughout), using PCR catalyzed by Pfu DNA polymerase (Stratagene, La Jolla, CA). The PCR product was initially blunt-end-ligated into pCR-Script using a commercial cloning kit (Stratagene). PCR primers contained flanking EcoRI and XbaI restriction endonuclease sites, enabling transfer of the PCR product into unique sites on the alpha 1C construct. The resulting chimeric channel contained amino acids 1-746 from alpha 1C, followed by 675-2213 from alpha 1A. To construct alpha 1AACC, we PCR-amplified a region spanning repeats III and IV of alpha 1C (nucleotides 3198-5193). PCR primers contained flanking AccI and XbaI restriction sites, permitting ready transfer of the PCR product into these sites on the alpha 1A construct. The resulting construct contained amino acids 1-1335 of alpha 1A, followed by 1067-1732 of alpha 1C and a premature stop codon. The premature stop codon was designed so as to delete the last 439 amino acids of the alpha 1C carboxyl tail because it has been previously demonstrated that such truncation significantly enhances expression of current without an appreciable change in pharmacological profile (29).


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  		Fig. 8.   Diltiazem inhibition of chimeric Ca2+ channels, derived from alpha 1C and alpha 1A. Left, data for alpha 1-CCAA. Top left, chimera diagrammed (dark portions are derived from alpha 1A). Right, results for alpha 1-AACC. Top right, diagram. A and B (top), exemplar current traces (open circle ) before and (bullet ) during exposure to 200 µM diltiazem. A and B (bottom), diary plots of peak current (open circle ) before, (bullet ) during, and (cross-centered circle) after exposure to 200 µM diltiazem. Arrows, traces shown above. C and D, diary plots of r100, the fraction of peak current remaining at the end of 100-msec depolarization. These demonstrate that diltiazem produced little change in current decay of (C) alpha 1-CCAA, in contrast to obvious acceleration of current decay of (D) alpha 1-AACC. C, For alpha 1-CCAA, the average change in r100 (drug - control) was small (-0.07 ± 0.04; three cells) and lacked statistical significance. D, In contrast, for alpha 1-AACC, the change in r100 was large (-0.26 ± 0.06; four cells) and significant (p < 0.05). Test depolarizations were delivered every 15 sec from a holding potential of -80 mV, with 30 mM Ba2+ as charge carrier. Both chimeric alpha 1 constructs were coexpressed with beta 2 and alpha 2 subunits.

We generated a mutant alpha 1C construct in which three amino acids in the IVS6 region were changed (Y1490I, A1494S, I1497M, numbering relative to alpha 1C) to those found in analogous positions of alpha 1A, yielding the mutant construct alpha 1C-ISM. To facilitate mutagenesis, two silent mutation were introduced into the standard alpha 1C construct (C4974T and G4977A), yielding a unique SfuI restriction site at nucleotide 4973. In the resulting alpha 1CSfuI+ construct, the IVS6 region was now bracketed by unique EcoRV and SfuI restriction sites, which are separated by only 626 base pairs. To generate alpha 1C-ISM, we used PCR mutagenesis by overlap extension (30). The outside primers contained EcoRV and SfuI restriction sites, permitting transfer of the PCR product (containing the desired mutations) into these sites on alpha 1CSfuI+. Portions of chimeric and mutant channel constructs derived from PCR were verified in their entirety with the use of the fluorescent dideoxy terminator method of thermocycle sequencing on an automated DNA sequencer (Applied Biosystems Division 373a; Perkin-Elmer Cetus, Norwalk, CT)

Electrophysiology. Whole-cell recordings were conducted at room temperature 1-3 days after transfection. The external solution contained 150 mM N-methyl-D-glucamine aspartate, 10 mM glucose, 10 mM HEPES, 10 mM 4-aminopyridine, and 0.1 mM EGTA, pH 7.3-7.4 with 1 M N-methyl-D-glucamine aspartate; 2-30 mM CaCl2 or BaCl2 was added as charge carrier. The internal solution contained 135 mM Cs-methanesulfonate, 5 mM CsCl, 10 mM EGTA, 10 mM HEPES, 1 mM MgCl2, and 4 mM MgATP, pH 7.2-7.3 with CsOH.

L-type Ca2+ channel antagonists, verapamil (Calbiochem, La Jolla, CA), and diltiazem (Sigma Chemical, St. Louis, MO), were dissolved in distilled water to make stock solutions (10 mM or 1 M) that were stored at -20°. Aliquots were diluted to the external solution to obtain the final desired concentrations.

For convenience, both Ca2+ and Ba2+ were used as charge carriers. Although an earlier study reported that the potency of Ca2+ channel blockers was modulated by the extracellular concentration and species of charge carrier (31), we found no evidence for such modulatory effects on alpha 1C and alpha 1A channels with the use of verapamil and diltiazem. We found that neither concentration (Ca2+, 5 versus 30 mM) nor charge species (5 mM Ca2+ versus 5 mM Ba2+, or 30 mM Ca2+ versus 30 mM Ba2+) significantly altered the extent of blockade by verapamil or diltiazem. Similar results were reported in the study of dihydropyridines (32, 33).

Transfected HEK 293 cells were grown onto coverslips, which facilitated easy transfer to a recording chamber just before electrophysiological recording. Fresh external solution continuously perfused the chamber at a flow rate of 1-2 ml/min. The bath was grounded by a 0.5 M KCl agar bridge attached to a Ag-AgCl wire. Whole-cell current records were obtained by standard patch-clamp techniques. Series resistance was typically < 5 MOmega and compensated > 60%. Leak and capacity transients were assessed between each test depolarization by a P/8 protocol and subtracted from test currents in all subsequent data analysis. Currents were filtered at 2 kHz (-3 dB, four-pole Bessel), sampled at 10 kHz, and stored digitally for data analysis.

Statistical analysis. All pooled data are reported as mean ± standard error. Statistical significance was assessed by two-sided, paired t test, with p < 0.05 taken as the minimal level of significance. N.S. denotes a comparison with no statistical significance (p > 0.05).

    Results
Summary
Introduction
Materials & Methods
Results
Discussion
References

Verapamil and diltiazem inhibition of multiple classes of Ca2+ channels. Although verapamil was developed as a blocker of L-type Ca2+ channels, Fig. 1 illustrates that the identical concentration of this compound (50 µM) produced significant block of all three classes of channels tested. The pharmacological profiles of alpha 1C, alpha 1A, and alpha 1E are presented in three columns, as labeled. The exemplar currents at the top, taken before and during application of verapamil (Fig. 1, A-C), explicitly demonstrate the inhibition of each type of channel. The diary plots of peak current shown below indicate the rapid inhibition of current on exposure to verapamil (bullet ), which is readily reversed on wash with control solution (triangle ), except in the case of alpha 1C. Slow or incomplete reversal of block was characteristic of alpha 1C.


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  		Fig. 1.   Verapamil inhibition of multiple classes of Ca2+ channels. Top (A-C), exemplar current traces from (A) alpha 1C, (B) alpha 1A, and (C) alpha 1E, all coexpressed with beta 2, in the (open circle ) absence and (bullet ) presence of 50 µM verapamil. The charge carrier was 5 mM Ca2+, and currents were evoked every 15 sec by 100-msec depolarizing pulses from a holding potential of -80 mV. Bottom (A-C), diary plots of peak current (open circle ) before, (bullet ) during, and (triangle ) after exposure to 50 µM verapamil. Arrows, traces shown above. D-F, Diary plots of r100, the fraction of peak current remaining at the end of 100-msec depolarization. Plots quantify the enhancement of current decay during verapamil inhibition. Results for alpha 1C, alpha 1A, and alpha 1E are representative of those observed in a total of seven, four, and five cells, respectively.

Apart from inhibition of peak current, a notable effect of verapamil was to accelerate the decay of current during maintained step depolarization. This effect is apparent in the exemplar traces (top, A-C), as well as in the diary plots of the fraction of peak current remaining at the end of 100-msec depolarizing steps (Fig. 1, D-F, r100). This acceleration of the decay of current is consistent with a state-dependent blocking mechanism in which verapamil preferentially inhibits the channel when it resides in or near the open and/or inactivated state. Such preferential inhibition of open/inactivated states could underlie the substantial use-dependent component of block observed in all channels with our 1/15 Hz stimulation frequency (data not shown). Overall, there was no fundamental difference in the action of verapamil on the three classes of channels.

In contrast, diltiazem produced fundamentally different effects on alpha 1C compared with alpha 1E or alpha 1A channels. Fig. 2 summarizes the pharmacological profile of diltiazem, following the same format as in Fig. 1. Although peak currents were readily inhibited by 100 µM diltiazem for all three channels (Fig. 2, A-C), the acceleration of current decay during maintained depolarizing steps was present in only alpha 1C. The latter finding is demonstrated by the exemplar records (top, A-C), as well as in the diary plots of r100 (D-F). The flat r100 plots for alpha 1A (Fig. 2E) and alpha 1E (Fig. 2F) quantitatively demonstrate the lack of change in current waveform with diltiazem exposure. These results suggested that diltiazem might selectively demonstrate preferential open/inactivated-state block of alpha 1C but not alpha 1A and alpha 1E channels. This idea was consistent with the absence of appreciable use-dependent block with alpha 1A and alpha 1E channels (data not shown). If confirmed, such a clearcut distinction in fundamental action could permit structural analysis of the basis of state-dependent block by diltiazem.


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  		Fig. 2.   Diltiazem inhibition of multiple classes of Ca2+ channels. The format is identical to that in Fig. 1, except that channels were inhibited by 100 µM diltiazem. Results for (A) alpha 1C, (B) alpha 1A, and (C) alpha 1E are representative of those observed in a total of four, four, and seven cells, respectively.

Dose-response curves for verapamil and diltiazem block. As another potential test for state-dependent block in a given drug/channel combination, we characterized the dose dependence of blockade by verapamil and diltiazem for all three types of channels. If a drug binds with different affinity to channels in distinct conformations, then the dose-response curve for drug blockade might be described by multiple binding isotherms corresponding to various channel states. On the other hand, if a drug binds with the same affinity, regardless of channel state, then the dose-response relation should describe a single Langmuir function.

Fig. 3 shows the dose-response of verapamil and diltiazem blockade for the three types of Ca2+ channels. In each case of verapamil blockade, fits of relations with single binding isotherms (not shown) consistently underestimated the degree of block in the 1-10 µM range, with differences between data and theoretical predictions ("residuals") ranging between 0.1 and 0.2. In contrast, fits with functions composed of two binding isotherms (solid gray curves) yielded no such consistent deviations in residuals, as if channels were roughly distributed among two groups of states: one with a high affinity for verapamil and the other with low affinity. These results fit nicely with the consistent acceleration of current decay produced by verapamil on all three classes of channels (Fig. 1). Therefore, both dose-response (Fig. 3A) and r100 data (Fig. 1, D-F) were consistent with state-dependent blockade of all three channels by verapamil.


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  		Fig. 3.   Concentration dependence of verapamil and diltiazem inhibition. Ordinate, peak current during steady state exposure to the blockers, normalized by the peak current before drug exposure, defined as Idrug/Ictrl. Points, mean ± standard error obtained from three to five cells. We applied only one concentration of drug to each cell, and the extent of drug block was measured as soon as peak currents showed no visible change from one pulse to the next. We thereby minimized possible distortion that might have occurred with a cumulative dose-response protocol, in which variable rundown could complicate interpretation. A, In each case of verapamil blockade, data were fit by a dual binding-site relation: Idrug/Ictrl = 1 - f1*[drug]/([drug] + Kd1) - f2*[drug]/([drug] + Kd2), where f1 + f2 = 1, and f1 and f2 represent the fraction of channels in high and low affinity binding conformations, respectively; and Kd1 and Kd2 represent dissociation constants for high and low affinity states, respectively. Dose-response relations for channels with high and low affinity states would be described by this two-isotherm relation as long as equilibration of drug binding is rapid relative to exchange between high and low affinity states. Solid gray curve, fit of this relation. Dashed and dotted curves, contribution of individual binding isotherms. A computer-based, iterative, nonlinear fitting algorithm was used. For alpha 1C, Kd1 = 0.29 µM, Kd2 = 220 µM, f1 = 0.19, and f2 = 0.81. For alpha 1A, Kd1 = 1.57 µM, Kd2 = 124 µM, f1 = 0.21, and f2 = 0.79. For alpha 1E, Kd1 = 0.13 µM, Kd2 = 123 µM, f1 = 0.09, and f2 = 0.91. When fits were performed with a one binding isotherm, there was a consistent deviation in residuals (theoretical prediction-data ~0.1 to 0.2) in the 1-10 µM range for all three channels. We observed no such consistent deviation in residuals with fits of the two-binding-isotherm function. For all data, currents were elicited by depolarizations to +10 mV, delivered every 15 sec from a holding potential of -80 mV, with 2 mM Ca2+ as charge carrier. B, Concentration dependence of diltiazem inhibition. The format is identical to that in A, except that single-binding isotherms were used to fit the data for alpha 1A and alpha 1E (Idrug/Ictrl = Kd1/([drug] + Kd1)). The fit parameters were as follows. For alpha 1C, Kd1 = 4.48 µM, Kd2 = 129 µM, f1 = 0.29, and f2 = 0.71. For alpha 1A, Kd1 = 270 µM. For alpha 1E, Kd1 = 220 µM. In the case of alpha 1C, the fit with a single-binding isotherm function consistently showed a residual theoretical prediction-data) averaging -0.15 at 10 µM diltiazem. No such deviation in residuals was observed with two-binding-isotherm fit shown (top, solid gray curve). For alpha 1A and alpha 1E, fits of a single binding isotherm (solid gray curves, middle and bottom, respectively) showed no consistent deviation of residuals.

The form of dose-response relations for diltiazem inhibition also agreed with our initial assignment of state-dependent block to alpha 1C (Fig. 2D), and state-independent block to alpha 1A (Fig. 2E) and alpha 1E (Fig. 2F). The alpha 1C data were well fit by a relation with two binding isotherms (Fig. 3B, top), which is consistent with the channel being distributed between higher and lower affinity states. Fits of relations with one binding isotherm (not shown) yielded consistent deviations in residuals, as observed above with verapamil. In contrast, the alpha 1A and alpha 1E dose-response data were well fit by a single binding term (Fig. 3B, middle and bottom), as if diltiazem had an equal affinity for all states.

Holding-potential dependence of drug inhibition. Another critical test for state-dependent block in certain drug/channel combinations is to determine whether the degree of drug inhibition is dependent on the holding potential between voltage pulses (32, 34). So far, all of our experiments were conducted with a fixed holding potential of -80 mV. At different holding potentials, the distribution of channels among various states was likely to be different. Consequently, if a drug bound with different affinity to different states, then the degree of inhibition would be affected by holding potential. Alternatively, if the drug bound with equal affinity to all states, no such holding potential dependence would be observed.

Fig. 4 shows the effects of holding potential on verapamil inhibition. The schematic (top) represents the holding potentials used in the various phases of the protocol. Brief test pulses were delivered every 15 sec from the indicated holding potentials. Because all measurements were taken over a short time period, we minimized the effects of drift and rundown. Before the application of verapamil, the holding potential was initially set at a depolarized level (-60 mV for alpha 1C and alpha 1A; -80 mV for alpha 1E), where the channel populated deep resting, shallow resting, and inactivated states. After stabilization at this potential (open circle ), we set the holding potential to a hyperpolarized value (-100 mV for alpha 1C and alpha 1A; -110 mV for alpha 1E) while still in the absence of verapamil. After equilibration (triangle ), inactivation was minimal, and most channels were in a deep resting state. The control currents obtained at these two potentials served to normalize currents inhibited by verapamil. While maintaining the hyperpolarized holding potential, verapamil was then applied, and the steady state level of inhibition was allowed to develop (black-triangle). The holding potential was then adjusted to the initial depolarized level and the steady level of inhibition allowed to develop with verapamil still present (bullet ). Verapamil was then removed, and the current allowed to recover at the depolarized (cross-centered circle) and hyperpolarized (cross-centered triangle) holding potentials. The degree of inhibition at the two holding potentials was obtained by normalizing inhibited currents by their corresponding control currents (hyperpolarized inhibition = black-triangle div  triangle ; depolarized inhibition = bullet  div  open circle ).


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  		Fig. 4.   Effect of holding potential on verapamil inhibition. Top, diagram of holding potentials used in the protocol described below. A-C, Diary plots of peak current measured during various phases of protocol. D-F, Extent of verapamil inhibition at different holding potentials for (D) alpha 1C, (E) alpha 1A, and (F) alpha 1E. Ordinate, fraction of control current remaining in the presence of verapamil. Statistical significance was from n cells as indicated. Brief test pulses to +10 mV were delivered every 15 sec from the indicated holding potentials, with 5 mM Ca2+ as charge carrier. Before the application of verapamil, we set the holding potential to a depolarized level, where the channel populated deep resting, shallow resting, and inactivated states. After stabilization at this potential (open circle ), we changed the holding potential to a hyperpolarized value while still in the absence of verapamil. After equilibration (triangle ), inactivation was minimized, and most channels populated deep resting states. The control currents obtained at these two potentials served to normalize currents inhibited by verapamil. While maintaining the hyperpolarized holding potential, verapamil was applied, and the steady state level of inhibition was allowed to develop (black-triangle). The holding potential was then adjusted to the initial depolarized level, and the steady level of inhibition was allowed to develop with verapamil still present (bullet ). Verapamil was then removed, and the current was allowed to recover at the depolarized (cross-centered circle) and hyperpolarized (cross-centered triangle) holding potentials. The degree of inhibition at the two holding potentials was obtained by normalizing inhibited currents by their corresponding control currents (hyperpolarized inhibition = black-triangle div  triangle ; depolarized inhibition = bullet  div  open circle ). We chose verapamil concentrations of (B) 30 µM for alpha 1A, (A) 50 µM for alpha 1C, and (C) 100 µM for alpha 1E, at which comparable degrees of inhibition were produced at holding potentials of -110 to -100 mV.   

Results of this experiment for each of three classes of channels are reported in separate columns in Fig. 4, as labeled. At the top of each column (A-C) is a diary plot of peak current. Shown below is the degree of inhibition at hyperpolarized and depolarized holding potentials. For each channel type, inhibition was clearly greater at the depolarized holding potential, which is consistent with state-dependent block of all channels by verapamil.

Fig. 5 summarizes results for the same holding potential experiment with diltiazem, using the identical format. Here, only alpha 1C showed greater inhibition at the depolarized holding potential (Fig. 5A); alpha 1A and alpha 1E were inhibited to the same extent (Figs. 5, B and C), regardless of holding potential. These results support the view that only alpha 1C showed state-dependent block by diltiazem.


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  		Fig. 5.   Effect of holding potential on diltiazem inhibition. The format and protocol are identical to those in Fig. 4, except that 100 µM diltiazem was used for drug inhibition throughout.

Unequivocal support for the results of these two-point holding potential experiments came with quantitative shape analysis of complete steady state inactivation (hinfinity ) curves, obtained in the presence and absence of drug. Following the same rationale as the streamlined holding potential protocol above, state-dependent blockade should not only depress the hinfinity curve but also shift its position after renormalization. State-independent block should depress the hinfinity curve without a shift in position. Fig. 6 shows the results of such an experiment, in which hinfinity curves were obtained with 20-sec prepulses to various voltages. Data for the three classes of channels are presented in separate columns, as labeled. At the top are shown hinfinity curves obtained in the absence (open symbol) and presence (filled symbol) of verapamil. In each case, there was a substantial depression of hinfinity curves by verapamil. At the bottom are shown two hinfinity curves after normalization to facilitate shape comparison. Clearly, there were changes in shape with drug inhibition of all channels, which is consistent with state-dependent inhibition throughout. Fig. 7 summarizes identical experiments, now with diltiazem as the blocker. After normalization of hinfinity curves (bottom, A-C), only alpha 1C gave evidence of state-dependent block.


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  		Fig. 6.   Effect of verapamil on steady state inactivation (hinfinity ) curves. Data for three classes of channels are presented in separate columns as labeled. Top, hinfinity curves obtained in the (open) absence and (filled) presence of 50 µM verapamil. Ordinate, peak test currents after normalization by the peak test current obtained following a prepulse to -110 mV in the absence of drug. Bottom, two hinfinity curves, after normalization to facilitate shape comparison. All channel types show shifts along the voltage axis. Protocol, every 60 sec, test depolarizations to +10 mV were delivered after a 20-sec prepulse to voltages from -110 mV to 0 mV. The holding potential was -110 mV throughout. The charge carrier was 5 mM Ca2+. Separate groups of cells were averaged for (open ) control and (filled ) drug data. Each group contained four or five cells.


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  		Fig. 7.   Effect of diltiazem inhibition on steady state inactivation (hinfinity ) curves. Data for the three classes of channels are presented in separate columns as labeled. The format is identical to that in Fig. 6, except that 100 µM diltiazem was used for drug inhibition. After normalization to facilitate shape comparison (bottom row), shifts along the voltage axis are apparent for only alpha 1C; the normalized hinfinity relations for alpha 1A and alpha 1E essentially superimpose, arguing for state-independent block. Control and drug data were averaged from different groups of cells. The control (open) data are replotted from Fig. 6. The drug (filled) data are averaged from four or five cells in each group.

Structural determinants of state-dependent block by benzothiazepines. Taken together, the r100 (Figs. 1 and 2), dose-response (Fig. 3), and holding-potential (Figs. 4, 5, 6, 7) experiments provided good evidence that a benzothiazepine produced state-dependent block of only alpha 1C channels, not alpha 1A or alpha 1E channels. This clear distinction in a fundamental pharmacological property furnished us a novel opportunity to apply chimeric and mutant Ca2+ channel analysis to gain insight into the structural determinants of state-dependent block.

According to the guarded-receptor hypothesis of drug block, the drug-binding site and guard elements that control drug access to the binding site are physically distinct structures (18). Critical amino acids for diltiazem binding have been localized to IVS6 (12), with potential ancillary contribution from IIIS6 (13). We therefore wondered whether structures specifying properties of state dependence were located on repeats III and IV near the binding site or could be entirely distinct structures residing on repeats I and II. Accordingly, we examined diltiazem blockade of chimeric Ca2+ channels in which repeats I and II were interchanged between alpha 1C and alpha 1A. The resulting chimeric channels are schematized in Fig. 8 (top). The profile of diltiazem blockade for the two constructs appear in separate columns, as labeled. Both channel constructs were readily inhibited by 200 µM diltiazem, as demonstrated in Fig. 8, A and B, by the exemplar current records (top) and the diary plots of peak current (bottom). Interestingly, the recovery from inhibition was rapid in alpha 1CCAA but slow in alpha 1AACC, suggesting that the characteristic slow recovery of alpha 1C (Fig. 2A) was mediated by structures in repeats III and IV near the binding site. The most important new findings came with the effects of diltiazem on the decay rate of currents. Close inspection of the exemplar traces shows that diltiazem hardly affected the waveform of alpha 1CCAA (Fig. 8A, top) but significantly accelerated the decay of current of alpha 1AACC (Fig. 8B, top). The r100 diaries, shown below in Fig. 8, C and D, confirm these impressions, which argue for the predominance of repeats III and IV in mediating the state-dependent phenotype.

Such predominance of repeats III and IV gave reason to wonder whether the very amino acids that are critical for higher affinity diltiazem binding in alpha 1C (12) are also essential to the state-dependent phenotype. A tyrosine, an alanine, and an isoleucine located in IVS6 of alpha 1C (Y1490, A1494, and I1497 in the rabbit cardiac alpha 1C used here) have been proposed to be crucial to diltiazem inhibition; when the corresponding amino acids in alpha 1A are mutated to these amino acids, higher affinity diltiazem blockade is conferred to the alpha 1A backbone (12). Here, we performed the converse experiment and mutated the critical amino acids in alpha 1C to their alpha 1A counterparts. We then asked whether these amino acids alone, which are a critical part of the binding pocket, are essential to the state-dependent phenotype.

Fig. 9 shows the effects of diltiazem on the resulting construct, alpha 1C-ISM. The exemplar currents and diary plot (Fig. 9A) demonstrate the ready block of peak current by diltiazem, although the half-maximal inhibitory concentration was increased by ~10-fold (not shown). More telling was the effect of diltiazem on the decay of current in the exemplar records. In addition to the substantial inhibition of current, diltiazem still produced a marked acceleration of the current decay. This impression was confirmed by the obvious decline in the diary plot of r100 during diltiazem application (Fig. 9B, bottom). These results suggested that diltiazem still preferentially inhibited channels in the open and/or inactivated states.


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  		Fig. 9.   Diltiazem inhibition of alpha 1C-ISM, an alpha 1C construct in which three point mutations in IVS6 have been made to remove the high affinity coordinating sites for diltiazem (Y1490I, A1494S, I1497M). Top, location of the point mutations is diagrammed. A (top), exemplar current traces (open circle ) before and (bullet ) during exposure to 200 µM diltiazem. A (bottom), diary plots of peak current (open circle ) before, (bullet ) during, and (black-triangle) after exposure to 200 µM diltiazem. Arrows, traces shown above. B, Diary plot of r100, the fraction of peak current remaining at the end of 100-msec depolarization, corresponding to the same cell and protocol as in A. The depression of r100 with diltiazem demonstrates obvious speeding of current decay with drug application. For the exemplar experiment (A and B), 5 mM Ba2+ was the charge carrier. There was no difference in the change of r100 upon drug application with Ba2+ or Ca2+ serving as charge carrier. Overall, the change in r100 was large and reproducible, averaging -0.16 ± 0.02 (p < 0.01 for seven cells: three with 5 mM Ba2+ and four with 5 mM Ca2+ as charge carrier). C, Diary plot of peak current measured during various phases of holding potential (VH) protocol diagrammed (top). Protocol was identical to that in Fig. 5. D, Extent of diltiazem inhibition at different holding potentials, derived from diary plots as in C. Statistical significance was assessed from four cells, with 5 mM Ca2+ as charge carrier. alpha 1C-ISM was coexpressed with beta 2 and alpha 2 subunits throughout.

To bolster the idea that diltiazem block of alpha 1C-ISM was state dependent, we examined the holding-potential dependence of diltiazem blockade of alpha 1C-ISM. Fig. 9, C and D, summarizes the results of a two-point holding potential experiment that is identical in every regard to that reported for alpha 1C in Fig. 5, A and D. The data clearly argue that there was still an appreciable effect of holding potential on diltiazem blockade.

The experiments shown in Fig. 9 consistently demonstrate that there must be other distinct structural elements in repeats III and IV, different from the amino acids most critical to diltiazem binding (Y1490, A1494, I1497), that are essential for state-dependent blockade. This result has important structural implications on which we elaborate in the Discussion.

    Discussion
Summary
Introduction
Materials & Methods
Results
Discussion
References

We report on the first systematic characterization of phenylalkylamine and benzothiazepine inhibition of three molecular classes of Ca2+ channels (alpha 1A, alpha 1E, and alpha 1C), expressed individually from recombinant clones. Although phenylalkylamines and benzothiazepines were developed as blockers of L-type (alpha 1C) channels, we find that verapamil and diltiazem inhibit all three classes of channels tested, with half-maximal inhibitory concentrations that differ by less than several-fold (Fig. 3). There are striking qualitative differences in the state dependence of inhibition observed in different drug/channel combinations. Although verapamil shows evidence of state-dependent inhibition in all three channels, diltiazem produces state-dependent block of only alpha 1C channels (not alpha 1A and alpha 1E channels). The determination of state dependence is based on mutually consistent results from current decay (Figs. 1 and 2), dose-response (Fig. 3), and holding-potential (Figs. 4, 5, 6, 7) experiments. This is the first instance in which an organic Ca2+ channel blocker demonstrates similar overall potency in different channels but a fundamental difference in the state-dependent character of blockade. Verapamil, D888, and mibefradil all show signs of state-dependent blockade of several molecular classes of Ca2+ channels (9-11, 35).

Relation to previous studies. For channel/drug combinations that manifest state-dependent blockade, the extent of blockade will vary considerably depending on the holding potential (Figs. 6 and 7) and stimulation rate. Therefore, in comparing the half-maximal inhibitory concentrations specified in Fig. 3 with those in the literature (12), it is worth emphasizing that the data in these figures were obtained with a uniform holding potential and stimulation rate (-80 mV and 1/15 Hz, respectively). In instances in which there was considerable state-dependent blockade, the half-blocking concentration would certainly have been lower with depolarization of the holding potential and/or increase in the stimulation rate.

Our finding of selective state dependence of diltiazem blockade with alpha 1C (but not with alpha 1A and alpha 1E) contrasts somewhat with another study (12) in which recombinant alpha 1A currents, expressed in Xenopus laevis oocytes, showed evidence of state-dependent diltiazem blockade. The difference may arise from the use of different alpha 1A clones [rat brain in our case (24) versus rabbit brain in the other study (36)] or different beta  subunits [rat brain beta 2 in our case (20) versus rabbit skeletal beta 1a (37)]. In the former case, it may be that a restricted number of differences between the two alpha 1A clones account for the difference in state dependence, but it is interesting that our finding of state-independent diltiazem block extends to a construct as different as alpha 1E. Differences in pharmacological profile related to alternative splicing (38) looms as another possibility with enormous therapeutic potential. Further work is clearly necessary to characterize the structural determinants of the state-dependent phenotype.

Implications for drug discovery. The finding that diltiazem blocks only alpha 1C in a state-dependent manner gives reason to expect that there exist organic compounds that would demonstrate selective state-dependent blockade of certain neuronal Ca2+ channels. Our results with diltiazem argue that the general functional requirements of Ca2+ channels have not imposed common structural themes that require organic compounds to demonstrate similar state-dependent blocking properties across channels, as in the case of verapamil. In fact, eliprodil, an organic drug initially developed as an N-methyl-D-aspartate channel antagonist, seems to demonstrate selective blockade of N-type (alpha 1B) and P/Q-type (alpha 1A) channels while sparing L-type (alpha 1C) and R-type (alpha 1E) channels (39). There is great interest in discovering organic compounds with selectivity for neuronal channels because they may prove to be easier to obtain and deliver than neuropeptides, which despite their proven selectivity for particular types of neuronal channels, may have difficulty crossing the blood-brain barrier to reach targets in the central nervous system (35).

Molecular basis of state-dependent diltiazem blockade. Chimeric Ca2+ channel analysis between alpha 1C and alpha 1A localized most of the structural determinants for state-dependent diltiazem blockade to repeats III and IV of alpha 1C, which likely contain the elements required for benzothiazepine binding (12, 13). Analysis of alpha 1C-ISM, a construct in which the predominant structural determinants of higher-affinity diltiazem inhibition of alpha 1C have been changed to their alpha 1A counterparts, revealed that state-dependent diltiazem inhibition was spared, despite a ~10-fold increase in half-maximal inhibitory concentration relative to alpha 1C. These results suggest that although the elements responsible for state dependence ("guards") are largely contained within repeats III and IV, they may be structurally distinct from the most critical part of the actual binding region in IVS6. The existence of physically distinct elements controlling drug binding and access to the receptor fits naturally with a guarded-receptor (18) rather than a modulated receptor mechanism of drug inhibition (16, 17). Such a physical dissociation of elements controlling binding and access has also been demonstrated in voltage-gated Na+ channels (40). It will be interesting to see whether such a physical distinction turns out to be a general design theme for state-dependent block of all voltage-gated channels.

    Acknowledgments

We are grateful to Dr. Shao-kui Wei (Johns Hopkins University School of Medicine) for his collaboration on certain experiments, to Yan Wang for constructing alpha 1CSfuI+, and to Dr. Paul Fuchs, David Brody, Lisa Jones, and Parag Patil for discussion and comments. We acknowledge the following individuals for generously providing Ca2+ channel clones: Terry P. Snutch (University of British Columbia, Vancouver, British Columbia, Canada) (alpha 1A, alpha 1E, alpha 2), Edward Perez-Reyes (Loyola University, Chicago, IL) (alpha 1C, beta 2a), and the late Chris Wei (Georgia Medical College) (alpha 1C).

    Footnotes

Received December 11, 1996; Accepted February 13, 1997

   This work was supported by Specialized Center of Research in Sudden Cardiac Death Grant NIH P50-HL52307 (D.T.Y.) and Postdoctoral Training Fellowship NIH 5-T32-HL07581 (D.M.C.).

Send reprint requests to: David T. Yue, M.D., Ph.D., Program in Molecular and Cellular System Physiology, Department of Biomedical Engineering, Johns Hopkins University School of Medicine, 720 Rutland Avenue, Baltimore, MD 21205. E-mail: dyue{at}bme.jhu.edu

    Abbreviations

HEK, human embryonic kidney; PCR, polymerase chain reaction; EGTA, ethylene glycol bis(beta -aminoethyl ether)-N,N,N',N'-tetraacetic acid; HEPES, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid.

    References
Summary
Introduction
Materials & Methods
Results
Discussion
References

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