©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
Transfer of L-type Calcium Channel IVS6 Segment Increases Phenylalkylamine Sensitivity of (*)

(Received for publication, December 15, 1995; and in revised form, March 5, 1996)

Frank Döring (§) Vadim E. Degtiar (§) Manfred Grabner Jörg Striessnig Steffen Hering (¶) Hartmut Glossmann

From the Institut für Biochemische Pharmakologie, Peter Mayr Strabetae 1, A-6020 Innsbruck, Austria

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Conditioned (``use-dependent'') inhibition by phenylalkylamines (PAAs) is a characteristic property of L-type calcium (Ca) channels. To determine the structural elements of the PAA binding domain we transferred sequence stretches of the pore-forming regions of repeat III and/or IV from the skeletal muscle alpha(1) subunit (alpha) to the class A alpha(1) subunit (alpha) and expressed these chimeras together with beta and alpha(2)/ subunits in Xenopus oocytes. The corresponding barium currents (I) were tested for PAA sensitivity during trains of depolarizing test pulses (conditioned block). I of oocytes expressing the alpha subunit were only weakly inhibited by PAAs (less than 10% conditioned block of I during a 100-ms pulse train of 0.1 Hz). Transfer of the transmembrane segment IVS6 from alpha to alpha produced an enhancement of PAA sensitivity of the resulting alpha/alpha chimera comparable to L-type alpha(1) subunits (about 35% conditioned block of I during a 100-ms pulse train of 0.1 Hz). Our results demonstrate that substitution of 11 amino acids within the segment IVS6 of alpha with the corresponding residues of alpha is sufficient to transfer L-type PAA sensitivity into the low sensitive class A Ca channel.


INTRODUCTION

Voltage-gated Ca channels mediate the depolarization-induced influx of Ca into excitable cells, thereby regulating cellular processes such as muscle contraction, propagation of action potentials, secretion, and gene expression. They are heterooligomeric complexes formed by at least an alpha(1), beta, and alpha(2)/ subunit(1) . The alpha(1) subunit is the pore-forming membrane protein consisting of four homologous repeats (I-IV), each of them composed of six transmembrane segments (S1-S6)(2) . Based on different pharmacological and biophysical properties, various types of voltage-dependent Ca channels (T, L, N, P, Q, and R) can be distinguished (3, 4, 5) . Their sensitivity to Ca antagonists or toxins is determined by the alpha(1) subunit(4, 6) . At least six different alpha(1) subunit genes have been isolated so far (for nomenclature, see (3) ). The alpha(1) subunit classes C (alpha), D (alpha), and S (alpha) mediate the high affinity of L-type Ca channels toward Ca antagonists, such as 1,4-dihydropyridines (DHPs), (^1)benzothiazepines, and phenylalkylamines (PAAs)(6, 7) . In contrast, classes A, B, and E are considered to be DHP-insensitive.

To localize Ca antagonist interaction domains within L-type alpha(1) subunits we have recently shown (8) that sensitivity for DHP Ca channel blockers and activators can be transferred to class A alpha(1) subunits (alpha) by substituting regions close to the channel pore in repeats III and IV (segments IIIS5, IIIS6, IVS6, and the respective S5-S6 linkers) with the corresponding L-type alpha(1) sequences (from alpha or alpha). The DHP sensitivity was lost after replacement of short sequence stretches within these regions by the alpha sequence. For example, when segment IIIS5 was replaced by alpha sequence, DHP sensitivity disappeared. The same effect was observed after replacing the IVS5-IVS6 linker. These results suggest that the DHP molecules interact with multiple amino acid residues located within distant regions of the primary structure.

Hockerman et al.(9) recently identified three amino acid residues within segment IVS6 of a L-type Ca channel alpha subunit (Tyr-1463, Ala-1467, and Ile-1470, numbering according to alpha) (10) as critical determinants for high affinity block by PAAs. Mutation of these residues within alpha to non-L-type resulted in a decrease of PAA sensitivity.

In our present work we studied the importance of the IVS6 segment for the formation of PAA interaction domains by investigating whether this region also supports PAA sensitivity in a non-L-type sequence environment. We addressed this question by testing whether the characteristics of L-type channel block by PAAs can be transferred to alpha that forms a non-L-type Ca channel. We demonstrate that currents through alpha expressed in Xenopus oocytes are less sensitive to PAAs than L-type currents. Transfer of the skeletal muscle IVS6 segment into alpha resulted in a chimeric alpha(1) construct that displayed PAA sensitivity comparable to L-type currents. We therefore conclude that L-type IVS6 also supports PAA sensitivity in a non-L-type sequence environment.


EXPERIMENTAL PROCEDURES

Materials

The phenylalkylamines(-)-D888) ((-)-devapamil, (-)-desmethoxy/verapamil), (±)-D600 ((±)-gallopamil, (±)-methoxyverapamil), and (±)-emopamil were kindly provided by Dr. Traut (Knoll AG, Ludwigshafen, Germany).

Construction of Chimeric alpha(1) cDNAs

alpha(1) chimeras (AL21, AL22, and AL23; for nomenclature, see Fig. 1A) consisting of alpha from rabbit brain (BI-2) (11) and alpha from carp skeletal muscle (12) were constructed and inserted into the polyadenylating transcription plasmid pNKS2 (provided by O. Pongs). Polymerase chain reaction (PCR) was used to create common restriction sites by introducing silent cDNA mutations. Mutations were introduced into forward and/or reverse PCR primers or by the ``gene SOEing'' technique(13) . Amplification of cDNA by PCR (Thermocycler 60, Biomed) was performed with 35 cycles at low stringency (1 min at 94 °C, 30 s at 42 °C, 1.5 min at 72 °C) using proofreading Pfu-polymerase (Stratagene). Chimeras AL21, AL22, and AL23 were constructed as follows (PCR-generated restriction sites are indicated by asterisks). AL21 (amino acid numbers in parentheses): A(1-1723), S(1311-1437), A(1856-2424). The SfiI-ClaI* fragment (nucleotide numbers in parenthesis) of A(4296-4925) was ligated into the SfiI (4296A) and ClaI* (4925A) sites of the chimeric construct AL9-pNKS2(8) . AL22: A(1-1723), S(1311-1402), A(1821-2424). A BamHI* site at position 4303 (S) was created by ``gene SOEing'' in the KpnI*-BglII (5467A-6185A) fragment of chimeric construct AL12s(8) . This PCR product was ligated into the KpnI* (5467A) and BglII (6185A) sites of AL21. AL23: A(1-1791), S(1374-1402), A(1821-2424). KpnI*-BamHI* fragment of A(5467-5667) was ligated into the KpnI* (5467A) and BamHI* (4303S) sites of AL22. The construction of chimeras L(h), L(s), AL1, and AL4 was described previously(8, 14) . The correct nucleotide composition of the chimeras was verified by extensive restriction enzyme mapping and by cDNA sequencing with the dideoxy chain termination method(15) .


Figure 1: Phenylalkylamine sensitivity of alpha, L-type alpha(1) chimeras and alpha/alpha chimeras. A, schematic representation of the studied alpha(1) subunits. L-type chimeras L(h) and L(s) are composed of sequences from the carp skeletal muscle alpha (black transmembrane segments and bold lines) and of the cardiac alpha (white segments and thin lines). To transfer PAA sensitivity to the alpha (sequence is indicated as gray segments and thin lines) sequence stretches from L-type channel alpha were inserted into alpha, thus generating chimeras AL1 and AL21-23. Alternatively, PAA sensitivity of chimera L(s) was reduced by replacing repeat IV and the carboxyl terminus by alpha sequence (chimera AL4). B, comparison of the conditioned I block of the alpha(1) subunits as depicted in A by different phenylalkylamines. The block of I was measured as cumulative current inhibition (in percent) during 12 depolarizing pulses (100 or 800 ms) after a 3-min incubation of the Xenopus oocytes in either 50 µM(-)-D888, 100 µM (±)-D600, or 100 µM(-)-emopamil as indicated. Oocytes were depolarized to the peak potential of the current voltage relationship. Duration of test pulses during a train was either 100 ms (black bars) or 800 ms (gray bars). Drop in peak I during the pulse protocol under control conditions indicates an incomplete recovery of I from inactivation during the train. Bars represent the mean ± S.E. of 3-19 experiments; *), p < 0.05; *, p < 0.01.



Expression of alpha(1) Chimeras in Xenopus laevis Oocytes

Preparation of stage V-VI oocytes from X. laevis and injection of cRNA were described in detail elsewhere (14) . The capped run-off poly(A) cRNA transcripts from XbaI linearized cDNA templates were synthesized according to the procedures of Krieg and Melton(16) . alpha(1) cRNAs (15 ng/50 nl) were coinjected with approximately equimolar ratios of beta(17) and alpha(2)/ (18) subunit cRNAs.

Voltage Clamp Measurements

Ba inward currents (I) through voltage-gated Ca channels were measured between 2 and 7 days after injection of the oocytes (19) using the two-microelectrode voltage-clamp technique (Turbo Tec 01C, NPI-Electronic, Germany). Endogenous Ca channel currents were studied after injection of beta and alpha(2)/ subunit cRNAs. Endogenous currents were present only in a minority of the tested oocyte batches and reached levels of expression between 5 and 80 nA. Only oocytes displaying I that where at least 10 times larger than endogenous currents were used for further analysis. Voltage-recording and current-injecting microelectrodes were filled with 2.8 M CsCl, 0.2 M CsOH, 10 mM EGTA, 10 mM HEPES (pH 7.4) and had resistances of 0.3-2 megohm. Oocytes were injected 20-40 min before the voltage-clamp experiments with 50 nl of a 0.1 M 1,2-bis(2-aminophenoxy)ethane-N,N,N`,N`-tetraacetic acid solution to block endogenous Ca-activated Cl conductance (see (20) ). All experiments were carried out at room temperature in bath solution with the following composition (in mM): 40 Ba(OH)(2), 40 N-methyl-D-glucamine, 10 HEPES, 10 glucose (pH adjusted to 7.4 with methanesulfonic acid). The recording chamber (150-µl total volume) was continuously perfused at a flow rate of 1 ml/min with control- or drug-containing solutions. Leakage current correction was performed by using average values of scaled leakage currents elicited by a 10-mV hyperpolarizing voltage step. The pClamp software package (version 5.51, Axon Instruments, Inc.) was used for data acquisition and analysis. Data were filtered at 1 kHz, digitized at 1 kHz, and stored on a computer hard disk.

Estimation of Conditioned I Block by PAAs

Conditioned (``use-dependent'') block of I by the PAAs(-)-D888 and (±)-D600 was measured during trains of either 100- or 800-ms test pulses. Test pulses were applied after a 3-min equilibrium period in drug-containing solution at a standard frequency of 0.1 Hz. The conditioned channel block by PAAs was estimated as the inhibition of peak I after 12 depolarizing test pulses (Fig. 1B and Fig. 2). The holding potential was -80 mV and test potentials were applied to the peak potential of the current voltage relationship of the Ca channel constructs. Because of incomplete recovery from inactivation of I, some chimeras displayed a decay in peak I during the pulse trains in the absence of drug. To estimate the peak I decay under control conditions we applied similar test pulses in the absence of drug, which were preceded by a 3-min rest period (see Fig. 1B and Fig. 2). The peak I inhibition during the first pulse after a 3-min equilibration in the drug-containing solution was defined as ``resting-state dependent block.'' Data are given as ranges or mean ± S.E. Statistical significance of I block by PAA compared to current decay under control conditions was calculated according to unpaired Student's t test.


Figure 2: I recordings illustrating PAA block of alpha, L-type alpha(1) chimeras and alpha/alpha chimeras. A, I through chimeras L(h) and L(s) during trains of 12 consecutive depolarizing voltage steps applied at 0.1 Hz in absence (control, left column) and presence (right column) of 50 µM(-)-D888. The pulse lengths were 100 and 800 ms for L(h) and 800 ms for L(s). B, I through alpha and chimeric alpha/alpha constructs AL22 and AL23 during 100-ms test pulse trains applied at 0.1 Hz. Recordings in the absence (control, left column) and presence (right column) of 50 µM of(-)-D888 are illustrated. The decrease in I in chimeras AL22 and AL23 in the absence of drug is caused by incomplete recovery from inactivation between the applied test pulses (compare Fig. 1B). Currents were recorded during depolarizations to 10 mV (A) and 15 mV (B) from a holding potential of -80 mV.




RESULTS

PAA Effects on L-type Ca Channel Currents

To investigate if Xenopus oocytes are an appropriate expression system for studying PAA effects on Ca channels, we compared the PAA sensitivity of wild type alpha with the previously described L-type alpha(1) chimeras L(h) and L(s)(8, 14) after expression in oocytes. The injection of alpha(1) subunit cRNAs, together with beta and alpha(2)/ subunit cRNAs, resulted in expression of calcium channels with barium current (I) amplitudes exceeding those of endogenous currents at least 10-fold (see ``Experimental Procedures'').

Both L-type chimeras are sensitive to DHP Ca channel agonists and antagonists and have been characterized previously(8, 14, 21) . Chimera L(h) (Fig. 1A) corresponds to alpha(22) but with its NH(2) terminus replaced by the respective sequence from carp skeletal muscle (alpha) (12) to increase the yield of expression(21) . Chimera L(s) (Fig. 1A) corresponds to L(h) with repeats III and IV replaced by sequence of the carp alpha. L(s) was tested for PAA sensitivity because carp alpha sequence stretches were used for the construction of alpha/alpha chimeras (see below). As shown in Fig. 1B L(h) and L(s) were blocked by micromolar concentrations of PAAs. Resting-state dependent I inhibition of chimera L(h) after 3 min of incubation with 50 µM (-)-D888 (9 ± 2%, n = 9, see Fig. 2A) was small and indistinguishable from current run-down during a similar period in drug free solution (5.5 ± 1.6%, n = 10). As PAA action on L-type Ca channels is crucially dependent on channel activation (see (23) ), we estimated the sensitivity of the expressed alpha(1) chimeras for PAAs as cumulative I inhibition during a pulse train (see ``Experimental Procedures''). Fig. 1B and 2A illustrate the conditioned block of chimeras L(h) and L(s) induced by 50 µM (-)-D888 or 100 µM (±)-D600. Prolongation of the test pulse duration from 100 to 800 ms substantially increased the extent of block by(-)-D888 (from 30 to 47% for L(h) and 26 to 45% for L(s); Fig. 1B and Fig. 2A). PAA-induced block of L(h) and L(s) was accompanied by an acceleration in current decay (Fig. 2A) which was most prominent for the slowly inactivating chimera L(s). In the presence of drug I recovered by 61 ± 7% (mean for L(h), n = 6) from conditioned block during a 3-min rest at -80 mV.

The PAA (±)-emopamil exhibits 1-2 orders of magnitude lower affinity for the PAA binding domain of L-type Ca channels than (-)-D888 and (±)-D600(24) . Unlike these PAAs, (±)-emopamil (100 µM) did not induce conditioned block of I (shown for chimera L(s) in Fig. 1B). This suggests that the observed PAA effects are mediated by specific interaction with the PAA binding domain.

PAA Effects on Ca Channel Currents through alpha Subunits

In contrast to the L-type Ca channel alpha(1) chimeras, PAA-induced block of I for alpha was much less pronounced. Fig. 2B illustrates I recordings from an oocyte expressing alpha during trains of test pulses in the absence and presence of 50 µM(-)-D888. In the absence of drug I decreased by 4 ± 1% (n = 7) during a 100-ms pulse train. This decrease in peak current amplitude was more pronounced (10 ± 4%, n = 5) after increasing the pulse length to 800 ms (Fig. 1B) and presumably resulted from incomplete recovery of I from inactivation. 50 µM(-)-D888 (Fig. 1B and Fig. 2B) or 100 µM (±)-D600 (Fig. 1B) caused a small but significant additional conditioned block of alpha current during 100-ms pulse trains (corresponding to about 10% of the peak current value). I block by 50 µM(-)-D888 during a 800-ms pulse train was enhanced to 21 ± 4% (n = 5) (Fig. 1B). Taken together, these data demonstrate that Ca channels formed by alpha subunits are only weakly sensitive to PAAs compared to the L-type chimeras L(s) and L(h).

PAA Effects on Ca Channel Currents through alpha/alpha Subunits

To determine if the PAA sensitivity of L-type Ca channels can be transferred from an L-type Ca channel to alpha, we constructed a series of chimeras between alpha and L-type sequence (Fig. 1A). When repeat IV and the adjacent carboxyl terminus of the PAA-sensitive chimera L(s) were replaced by alpha sequence PAA sensitivity of the resulting chimera AL4 (Fig. 1A) was reduced to the level of the alpha subunit (Fig. 1B).

AL1 represents the first of four chimeras in which alpha sequence was introduced into alpha within repeats III and IV. It contains L-type sequences in the S5-S6 linkers and adjacent segments S6 in repeats III and IV (8) (Fig. 1A). A substantial fraction of I from chimera AL1 did not recover from inactivation between the 10-s interpulse interval of the train in the absence of PAAs. As shown for alpha (Fig. 2B) this resulted in a decrease in I amplitude during frequent depolarizations and was more pronounced if prolonged test pulses were applied (data not shown). This prevented the analysis of conditioned block during trains of pulses longer than 100 ms. During 100 ms pulse trains the PAA sensitivity of AL1 was comparable to constructs L(h) and L(s): 50 µM (-)-D888 induced a conditioned block of 24% (n = 4) beyond the peak current decay of I observed in the absence of drug (Fig. 1B). In chimera AL21 repeat III completely consisted of alpha sequence. The observed PAA sensitivity still resembled that of AL1 (Fig. 1B). Furthermore, neither the removal of L-type sequence on the cytoplasmic side of IVS6 (generating chimera AL22) nor of the IVS5-IVS6 linker (leading to chimera AL23, see Fig. 1and Fig. 2B) decreased the PAA sensitivity. I through chimera AL23 exhibited less than 2% (n = 12) run-down and displayed the characteristic features of PAA sensitivity. (i) Resting state-dependent I block of chimera AL23 was less than 5% (n = 12), (ii) the fraction of Ca channels blocked by PAAs was dependent on the application of depolarizing test pulses (I was inhibited during a train of 100-ms pulses by 57 ± 6% (n = 7) in the presence of 50 µM(-)-D888 compared to 21 ± 4% (n = 12) under control conditions; Fig. 1B), and (iii) I recovered from conditioned block in the presence of 50 µM(-)-D888 by 95 ± 3% (n = 12) during a 3-min rest at -80 mV. As was the case with L(h) and L(s), no frequency-dependent effect of (±)-emopamil was observed during trains of 100-ms test pulses in chimera AL23 (Fig. 1B).

Biophysical Properties of Chimera AL23

In 40 mM Ba solution I of chimera AL23 activated at a threshold of -10 mV and reached peak current values between 10 and 20 mV (16 ± 1.2 mV, n = 26). The I of alpha had a similar threshold as AL23 (-10 mV) and reached a peak current at 13 ± 1.4 mV (n = 15). However, AL23 (as well as AL21 and AL22) inactivated faster than alpha (Fig. 2B). I of AL23 decayed by 68 ± 6% (n = 12) during a 100-ms test pulse to 10 mV, whereas only 14 ± 4% (n = 10) of alpha current inactivated under identical experimental conditions. These data suggest a possible role of structural elements of segment IVS6 in inactivation of alpha.


DISCUSSION

To gain further insight into the molecular organization of the high affinity PAA binding domain, we investigated if the transmembrane domain IVS6 of a skeletal muscle alpha subunit (25) transfers L-type PAA sensitivity to a non-L-type channel alpha(1) subunit. The alpha subunit, as a putative PAA-insensitive alpha(1), was selected for the following reasons. (i) With the exception of alpha and alpha, all other alpha(1) subunits cloned so far (including alpha) (9) have been shown or are considered to be (3) PAA-sensitive to various degrees. (ii) We recently demonstrated that DHP sensitivity can be transferred to alpha by introducing L-type channel (alpha or alpha) sequences into regions surrounding the channel pore(8) . (iii) The segment IVS6 of alpha does not contain the high affinity PAA binding motif described by Hockerman et al.(9) .

We found that alpha, when coexpressed in Xenopus oocytes together with beta and alpha(2)/, exhibited low sensitivity to PAAs. This is in contrast to DHP sensitivity, which is absent in alpha(8, 26, 27) . The PAA sensitivity was, however, much smaller than that for L-type chimeras L(h) and L(s) (Fig. 1B). Analysis of PAA effects on several alpha/alpha chimeras revealed that introduction of L-type (alpha) IVS6 was sufficient to increase PAA sensitivity to L-type levels, whereas replacement of the entire repeat IV in the chimera L(s) by alpha sequence decreased PAA sensitivity. Fig. 3illustrates that segments IVS6 are largely conserved between L-type alpha and alpha and differ only in 11 positions. These include positions 1386, 1390, and 1393 of alpha (numbering according to Grabner et al.(12) ) that are known to comprise critical determinants of PAA sensitivity in L-type channels(9) . Our data demonstrate that segment IVS6 can participate in the formation of a PAA binding pocket in a non-L-type channel environment. The finding that the exchange of 11 amino acids residues within a single putative transmembrane helix (Fig. 3) is sufficient to increase alpha PAA sensitivity, differs from our previous observations with DHPs. To obtain a DHP agonist and antagonist sensitive chimera, L-type sequences had to be transferred into transmembrane segments IIIS5, IIIS6, and IVS6 as well as into the respective S5-S6 linkers(8) . In contrast, the transfer of PAA sensitivity from L-type Ca channels into alpha did not require the introduction of other than the IVS6 sequence. The low PAA sensitivity of alpha suggests that additional interaction sites are provided by alpha. Low PAA sensitivity was also observed for N-type Ca channels (alpha) and for L-type channels (alpha) lacking the high affinity determinants for PAA sensitivity in segment IVS6(9) . Therefore, additional regions of PAA interaction may be localized in sequence stretches conserved among these alpha(1) subunits. Future studies will concentrate on the possible involvement of these regions in the interaction of Ca channels with PAAs.


Figure 3: Sequence alignment of transmembrane segments IVS6 of skeletal and cardiac L-type and class A alpha(1) subunits. The sequence stretch of carp alpha(12) which was implanted into alpha leading to the PAA sensitive chimera AL23 is aligned with the corresponding regions of alpha(22) and of alpha (BI)(11) . Numbers of amino acids correspond to alpha. Residues within the alpha sequence different from the L-type alpha are highlighted. Asterisks indicate amino acids which were identified to be critical for PAA sensitivity in L-type Ca channels(9) .



As previously shown in mammalian cells, PAA block of I in Xenopus oocytes is also dependent on the application of depolarizing test pulses (Fig. 2). PAAs are believed to interact selectively with the open Ca channel conformation (28, 29) which complicates an estimation of drug association and dissociation rate constants(30) . Inhibition of open Ca channels by PAAs is also supported by the observed acceleration of I decay in chimeras L(h) and L(s) in the presence of PAAs (Fig. 2A). The extent of block induced by(-)-D888 (50 µM) and (±)-D600 (100 µM) during 12 pulses (100 ms) in chimera AL23 is comparable to the inhibition of the L-type chimeras L(h) and L(s). Resting state-dependent block was almost absent, but pronounced inhibition developed during a train of depolarizing pulses. Furthermore, blocked I recovered almost completely during a 3-min pulse-free interval in the presence of drug. Thus, the mechanism of Ca channel block in Xenopus oocytes appeared to be similar to PAA block of L-type channels in various mammalian cells(23, 28, 31) . As previously observed, e.g. for DHPs (8) and Ca antagonist Ro 40-5967 (32) , the effective drug concentrations for channel block after expression in Xenopus oocytes were higher than required in electrophysiological studies using mammalian cells (see Refs. 9 and 28).

An additional finding of our study was that introducing L-type sequence into alpha did not only enhance PAA sensitivity but also changed the inactivation kinetics of I. Interestingly, the implantation of the transmembrane segment IVS6 from the slowly inactivating L-type chimera L(S) into alpha did not result in a transfer of the slower L-type inactivation kinetics into the faster inactivating alpha. Unexpectedly, this sequence substitution accelerated the inactivation kinetics compared to that of alpha. This finding gives an example where kinetic properties of Ca channels are not simply transposed by swapping corresponding sequences between different alpha(1) subunits as was previously shown for structural elements of repeat I as well as III and IV(14, 33, 34) . Our observation, that inactivation of alpha Ca channels is accelerated by changes in the amino acid sequence of segment IVS6 indicates a possible involvement of this region in inactivation gating in addition to its role in forming the PAA interaction domain.


FOOTNOTES

*
This work was supported by grants from the Fonds zur Förderung der Wissenschaftlichen Forschung (S6601-med to H. G., S6602-med to J. S., and S6603-med to S. H.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Both authors contributed equally to this work.

To whom correspondence should be addressed. Tel.: 43-512-507-3154; Fax: 43-512-588627.

(^1)
The abbreviations used are: DHP, dihydropyridine; PAA, phenylalkylamine;(-)-D888, devapamil; (±)-D600, gallopamil; I, barium inward current through Ca channels; PCR, polymerase chain reaction.


ACKNOWLEDGEMENTS

We are grateful to Drs. Y. Mori and K. Imoto for the generous gift of the alpha cDNA, A. Schwartz for the alpha and alpha(2)/ cDNAs, O. Pongs for providing the transcription plasmid pNKS2, Dr. Z. Wang for experimental support, B. Kurka for expert technical assistance, and Dr. B. Flucher for critical comments on the manuscript. We also thank Dr. W. A. Catterall for preprints of unpublished work.


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