Nine L-type Amino Acid Residues Confer Full 1,4-Dihydropyridine Sensitivity to the Neuronal Calcium Channel α1ASubunit

Pharmacological modulation by 1,4-dihydropyridines is a central feature of L-type calcium channels. Recently, eight L-type amino acid residues in transmembrane segments IIIS5, IIIS6, and IVS6 of the calcium channel α1subunit were identified to substantially contribute to 1,4-dihydropyridine sensitivity. To determine whether these eight L-type residues (Thr1066, Gln1070, Ile1180, Ile1183, Tyr1490, Met1491, Ile1497, and Ile1498; α1C-a numbering) are sufficient to form a high affinity 1,4-dihydropyridine binding site in a non-L-type calcium channel, we transferred them to the 1,4-dihydropyridine-insensitive α1A subunit using site-directed mutagenesis. 1,4-Dihydropyridine agonist and antagonist modulation of barium inward currents mediated by the mutant α1A subunits, coexpressed with α2δ and β1a subunits inXenopus laevis oocytes, was investigated with the two-microelectrode voltage clamp technique. The resulting mutant α1A-DHPi displayed low sensitivity for 1,4-dihydropyridines. Analysis of the 1,4-dihydropyridine binding region of an ancestral L-type α1 subunit previously cloned from Musca domestica body wall muscle led to the identification of Met1188 (α1C-a numbering) as an additional critical constituent of the L-type 1,4-dihydropyridine binding domain. The introduction of this residue into α1A-DHPi restored full sensitivity for 1,4-dihydropyridines. It also transferred functional properties considered hallmarks of 1,4-dihydropyridine agonist and antagonist effects (i.e. stereoselectivity, voltage dependence of drug modulation, and agonist-induced shift in the voltage-dependence of activation). Our gain-of-function mutants provide an excellent model for future studies of the structure-activity relationship of 1,4-dihydropyridines to obtain critical structural information for the development of drugs for neuronal, non-L-type calcium channels.

Voltage-dependent calcium channels are activated by membrane depolarization and mediate the rapid and selective entry of calcium into excitable cells. The subsequent rise in intracellular calcium triggers a variety of cellular responses, including excitation-contraction coupling, excitation-secretion coupling, synaptic plasticity, and the modulation of transcription events (for a review, see Refs. 1 and 2). On the basis of electrophysiological and pharmacological criteria, voltage-dependent calcium channels are classified into L-, N-, T-, P/Q-, and R-type channels (reviewed in Refs. 3 and 4). The heterooligomeric channel complexes are composed of a pore-forming ␣ 1 subunit in combination with accessory subunits (␣ 2 ␦, ␤, and in skeletal muscle ␥), which modulate the pharmacological and kinetic channel properties (1,4). Molecular cloning and heterologous expression experiments have revealed that three classes of ␣ 1 subunits (␣ 1C (5) in heart, smooth muscle, and neurons; ␣ 1S (6) in skeletal muscle; and ␣ 1D (7) in neuroendocrine cells) form L-type calcium channels (3). They are distinguished from the other types by their high sensitivity to 1,4-dihydropyridines, phenylalkylamines, and benzothiazepines (8), which are used therapeutically for the treatment of a variety of cardiovascular disorders (9). 1,4-Dihydropyridine (DHP) 1 antagonists stabilize the inactivated state of the channel (10), whereas DHP agonists promote the open state (11). However, the molecular mechanism of channel modulation mediated by these drugs has yet to be completely elucidated.
The DHP binding domain is located on the ␣ 1 subunit (12). It is tightly coupled to a high affinity calcium binding site (13) representing the ion selectivity filter (14 -16). One essential requirement to fully understand the molecular mechanism of channel modulation is the identification of amino acid residues that mediate DHP agonist and antagonist effects. Photoaffinity labeling, combined with antibody mapping (12) as well as construction of chimeric ␣ 1 subunits (17) identified parts of the high affinity DHP binding domain. Using a gain-of-function approach, we have shown that introducing only as little as 9.4% L-type sequence (including transmembrane segments IIIS5, IIIS6, and IVS6) is sufficient to transfer DHP sensitivity to the DHP-insensitive class A (BI-2) calcium channel ␣ 1 subunit (18). Subsequently, we demonstrated that two amino acid residues of segment IIIS5 are critical for the DHP interaction (19). In transmembrane segments IIIS6 and IVS6 of the ␣ 1 subunit, six other L-type amino acid residues were identified to be required for DHP binding by creating chimeras and mutants that were monitored for a reduction or loss of DHP sensitivity (20,21).
Here we used a gain-of-function approach to investigate whether these eight L-type residues, when removed from their natural sequence environment, are able to form a functional * This work was supported by Fonds zur Förderung der Wissenschaftlichen Forschung Grants S6601 (to H. G.), S6602 (to J. S.), and S6603 (to S. H.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18  DHP interaction domain. The DHP-insensitive ␣ 1A subunit was chosen as the acceptor molecule (22,23,18). We discovered that an additional methionine residue in transmembrane segment IIIS6 of the L-type calcium channel is also required for DHP sensitivity. Together, these nine L-type amino acid residues of the resulting motif allow for the construction of a fully functional agonist-and antagonist-sensitive DHP binding pocket in ␣ 1A subunits.

EXPERIMENTAL PROCEDURES
Construction of Chimeric and Mutant ␣ 1 cDNAs-Class A/L-type chimeric ␣ 1 subunits derived from the DHP-insensitive rabbit brain class A calcium channel (BI-2) ␣ 1A (A; Ref. 22) and the Musca domestica (housefly) muscle ancestral L-type calcium channel ␣ 1M (M; Ref. 24) as well as mutants thereof were constructed by using the "gene SOEing" technique (25). "Silent" restriction endonuclease cleavage sites generated by polymerase chain reaction in previous cloning steps (18) are indicated by asterisks. Amino acid and nucleotide numbering is given in parenthesis.
For AL12m/V1048M, the single point mutation was introduced by using a HpaI-KpnI* cassette (nucleotides 3174M and 4023M, respectively) in AL12m.
Chimeras and mutants were inserted into the polyadenylating transcription plasmid pNKS2 (provided by O. Pongs, ZFMNB, Hamburg, Germany). cDNA amplification by polymerase chain reaction was performed as described previously (18). Mutagenic primers contained silent point mutations to introduce or eliminate restriction endonuclease sites for verification of the desired mutations. Fragments amplified by the polymerase chain reaction were sequenced entirely to verify sequence integrity (27).
Expression of ␣ 1 Chimeras and Mutants in Xenopus laevis Oocytes-Preparation of stage V and VI oocytes from Xenopus laevis and synthesis of capped run-off poly(A) ϩ cRNA transcripts from XbaI-linearized cDNA templates as well as coinjection of ␣ 1 cRNAs with ␤ 1a (28) and ␣ 2 ␦ (29) subunit cRNAs were described previously (18).
Voltage Clamp Measurements-Barium inward currents (I Ba ) through voltage-gated calcium channels were measured between 2 and 5 days after injection of cRNA using the two-microelectrode voltage clamp technique (Turbo Tec 01C, NPI-Electronic, Germany). Voltage-recording microelectrodes and current-injecting electrodes were filled with 2.8 M CsCl, 0.2 M CsOH, 10 mM EGTA, 10 mM HEPES (pH 7.4) and had resistances of 1-2 megaohms. All experiments were carried out at room temperature using barium as the charge carrier in a solution composed of 40 mM Ba(OH) 2 , 40 mM N-methyl-D-glucamine, 10 mM HEPES, and 10 mM glucose (pH adjusted to 7.4 with methanesulfonic acid). The recording chamber (volume, 150 l) was continuously perfused with a flow rate of 1 ml/min. Drug-containing solutions were applied with the same flow rate. Stock solutions (10 mM) of drugs were prepared in dimethyl sulfoxide and stored at Ϫ20°C in the dark. Experiments were carried out in sodium light, and stock solutions were freshly diluted in bath solution. Modulation of peak I Ba was measured from a holding potential of Ϫ80 mV in the absence (control) and presence of drug-containing solutions at equilibrium (half-time for the development of the stimulatory effect by 10 M (Ϯ)-BayK 8644 was 12 Ϯ 3 min, n ϭ 5, for ␣ 1A-DHP ). Test potentials corresponded to the peak potential of the current-voltage relations for measurements of I Ba block by antagonists, and 10 mV hyperpolarized to the peak potential of the current-voltage relations for measurements of agonist modulation. To obtain the voltage of half-maximal I Ba activation (V 0.5act ) current-voltage relation curves were fitted to the function where G max is the maximal conductance, V is the test potential, V rev is the reversal potential, and k act is the slope factor of the activation curve. I Ba inhibition (antagonist effects), given as percentage of inhibition, was calculated as (1 Ϫ peak I Ba (drug)/peak I Ba (control)) ϫ 100%, and current amplification (agonist effects) was calculated as peak I Ba (drug)/peak I Ba (control) and given as -fold stimulation. DHP effects were indistinguishable at pulse frequencies between 0.017 Hz and 0.1 Hz. Therefore, unless stated otherwise, a standard pulse frequency of 0.017 Hz and 0.034 Hz was used to assess agonist and antagonist modulation, respectively. 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 0.2 kHz, and stored on a computer hard disk. Benzoxadiazol-DHPs were from Sandoz AG (Basel, Switzerland), (Ϯ)-BayK 8644 was from Bayer AG (Wuppertal, Germany), and FPL 64176 was from Fisons Pharmaceuticals (Leicestershire, UK).
Statistics-Data are given as the mean Ϯ S.E. from the indicated number of experiments. Statistical significance was calculated using the unpaired t test. IC 50 values for the concentration-response relationships were determined by fitting the experimental data to the general dose-response equation (30).

Transfer of Eight L-type Amino Acids Creates a Mutant ␣ 1A Subunit with Weak Sensitivity to the DHPs (Ϯ)-BayK 8644 and
(Ϯ)-Isradipine-Using site-directed mutagenesis, we transferred the L-type residues Thr 1066 , Gln 1070 , Ile 1180 , Ile 1183 , Tyr 1490 , Met 1491 , Ile 1497 , and Ile 1498 (numbering according to ␣ 1C-a ) to the ␣ 1A subunit by replacing the corresponding non-L-type residues. The resulting mutant ␣ 1A-DHPi ( Fig. 1A) was coexpressed with auxiliary ␣ 2 ␦ and ␤ 1a subunits in Xenopus oocytes to test if these residues can form an L-type DHP binding domain within the background of ␣ 1A sequence. ␣ 1A-DHPi gave rise to barium inward currents with a threshold of activation between Ϫ35 and Ϫ25 mV (Fig. 1B). The midpoint voltage of current activation was Ϫ8.6 Ϯ 1.0 mV (n ϭ 13), and maximal I Ba amplitudes were reached at test potentials between 0 and 10 mV. I Ba modulation by the standard DHP probes (Ϯ)-BayK 8644 (agonist) and (Ϯ)-isradipine (antagonist) was considerably reduced when compared with recombinant L-type calcium channels expressed in Xenopus oocytes (18). Peak I Ba of ␣ 1A-DHPi , measured at peak potentials of the current-voltage relations, was blocked by (Ϯ)-isradipine (Fig. 1B, upper panel) with a low apparent potency, yielding an IC 50 of ϳ10 M (Fig. 4B). The stimulation of peak I Ba by 10 M (Ϯ)-BayK 8644 was only 1.6 Ϯ 0.1-fold (n ϭ 7, Fig. 4A) at test potentials 10 mV negative to the peak of the current-voltage relations (Fig. 1B, lower panel). Most features of BayK 8644 modulation were not detectable, such as the agonist-induced slowing of macroscopic I Ba -activation at threshold potentials (not shown) as well as a slowing of channel deactivation (Fig.  1B, lower panel). 10 M (Ϯ)-BayK 8644 failed to significantly (p Ͼ 0.01) shift the midpoint voltage of I Ba activation (V 0.5act ϭ Ϫ7.2 Ϯ 0.7 mV, n ϭ 3) but clearly elicited a change of macroscopic I Ba inactivation (Fig. 1B, lower panel).
The DHP antagonist effect on ␣ 1A-DHPi was stereospecific as expected for L-type channels. At identical concentrations (10 M) the plus enantiomer of isradipine was more potent than its minus counterpart (Fig. 1C). This finding suggested a specific interaction of isradipine with ␣ 1A-DHPi , albeit at a reduced sensitivity. Taken together, these data indicated that DHP sensitivity of ␣ 1A-DHPi is reduced when compared with recombinant L-type calcium channel ␣ 1 subunits coexpressed with ␣ 2 ␦ and ␤ 1a subunits in Xenopus oocytes (18,19). Thus, one or more other L-type amino acid residues are required for certain DHP actions and high DHP sensitivity.
Chimera AL12m Reveals the Importance of a Methionine Residue in IIIS6 for DHP Sensitivity-Recently, we have cloned the ancestral L-type ␣ 1M subunit from housefly (M. domestica) body wall muscle (24). Similar to L-type ␣ 1 subunits derived from vertebrate skeletal muscle (6,31), functional expression of ␣ 1M in Xenopus oocytes failed (24). We therefore attempted to investigate the DHP sensitivity of ␣ 1M by means of a chimeric calcium channel ␣ 1 subunit, termed AL12m ( Fig. 2A), that was constructed in analogy to the DHP agonist-and antagonistsensitive chimeras AL12h and AL12s (18). As shown in Fig. 2B, the I Ba -modulation of AL12m differed from AL12h and AL12s. I Ba of AL12m was clearly blocked by (Ϯ)-isradipine, but stimulation by (Ϯ)-BayK 8644 could not be detected.
We exploited this absence of agonist sensitivity to identify additional amino acid residues that are involved in the DHP interaction. Sequence alignments of transmembrane segments IIIS6 and IVS6 from cardiac and skeletal muscle L-type ␣ 1 subunits with ␣ 1M revealed three amino acid divergences (Fig.  2C) in ␣ 1M , located within previously described core regions of the DHP interaction domain (20,21). For the Musca channel, two Met to Val exchanges were identified, one each in IIIS6 and IVS6, and an Ala to Ser conversion was also found in IVS6. Interestingly, each of these residues (Met 1188 , Met 1491 , and Ala 1494 ; ␣ 1C-a numbering) is highly conserved among vertebrate L-type channel ␣ 1 subunits and possesses a highly conserved counterpart (Val 1512 , Phe 1805 , and Ser 1808 , respectively; ␣ 1A numbering) in non-L-type ␣ 1 subunits. We therefore converted single amino acid residues in AL12m to vertebrate Ltype sequence and tested the DHP sensitivity of the resulting chimeras AL12m/V1048M, AL12m/V1352M, and AL12m/ S1355A (numbering according to ␣ 1M ; Ref. 24). Both Val to Met conversions (but not the Ser to Ala substitution) resulted in an increased agonist sensitivity (Fig. 2E). In contrast to AL12mmediated currents, 10 M (Ϯ)-BayK 8644 stimulated the I Ba peak amplitudes of AL12m/V1048M and AL12m/V1352M 1.6 Ϯ 0.1-fold (n ϭ 4), and 1.4 Ϯ 0.1-fold (n ϭ 4), respectively. Although BayK 8644-mediated agonist modulation of these AL12m mutants did not reach the extent observed for recombinant L-type channels or the chimeras AL12s and AL12h (18), each of the methionine residues played an important role for agonist sensitivity. It remains to be investigated whether simultaneous conversion of both residues could elicit full BayK 8644 sensitivity. The inhibition of peak I Ba by 10 M (Ϯ)isradipine was 70 Ϯ 6% (n ϭ 5), 69 Ϯ 7% (n ϭ 4), 80 Ϯ 3% (n ϭ 3), and 64 Ϯ 6% (n ϭ 4) for AL12m, AL12m/V1048M, AL12m/ V1352M, and AL12m/S1355A, respectively (Fig. 2D), and therefore comparable (p Ͻ 0.01) for AL12m and the single mutants derived thereof. These findings suggested that these two methionine residues play a prominent role in agonist modulation, at least within a sequence environment that presumably contains additional, low affinity interaction sites for DHP antagonists in the S5-S6 linker of domain IV, similar to the ␣ 1S /␣ 1A -chimeras AL10 and AL13 (18).
The substantial contribution of Met 1491 (AL12m/V1352M) in transmembrane segment IVS6 to agonist sensitivity is in agreement with previously published studies (20,21). In contrast, the importance of Met 1188 (numbering according to ␣ 1C-a ) in IIIS6 for DHP agonist modulation represents an entirely new finding. Of these two L-type residues, only the methionine in IVS6 was present in ␣ 1A-DHPi . Consequently, the absence of Met 1188 could be responsible for the lower DHP sensitivity and an absence of both an agonist-induced shift in activation and slowing of channel deactivation of this mutant. To test this possibility, we introduced Met 1188 into ␣ 1A-DHPi . The resulting mutant was termed ␣ 1A-DHP .
Met 1188 Is Required for Full Agonist and Antagonist Sensi- Peak I Ba is plotted as a function of test potential in the current-voltage relations of the right column. Asterisks indicate the test potential of the traces depicted in the left column. The holding potential was Ϫ80 mV, and pulse duration was 350 ms. C, stereoselective inhibition of ␣ 1A-DHPi by 10 M solutions of the stereoisomers of isradipine. Currents were recorded after depolarization from a holding potential of Ϫ80 mV to a test potential of 10 mV. tivity in the mutant ␣ 1A Subunit- Fig. 3A shows a schematic drawing of ␣ 1A-DHP with Met 1188 in the IIIS6 segment. Coexpression of ␣ 1A-DHP with ␣ 2 ␦ and ␤ 1a subunits resulted in barium inward currents similar to ␣ 1A-DHPi . The threshold of current activation was between Ϫ35 and Ϫ25 mV, and the half-activation potential of Ϫ10.3 Ϯ 1.3 mV (n ϭ 12) was indistinguishable (p Ͼ 0.01) from the midpoint voltage of current activation determined for ␣ 1A-DHPi . Maximal current amplitudes were also reached at test potentials between 0 and 10 mV (Fig. 3B). Macroscopic I Ba inactivation (expressed as percentage of peak current decay during 100 ms at test potentials corresponding to the peak of the current-voltage relations) was less in ␣ 1A-DHP (32 Ϯ 1%; n ϭ 12) than in ␣ 1A-DHPi (56 Ϯ 1%; n ϭ 13). Differences in the response of ␣ 1A-DHP compared with ␣ 1A-DHPi to the agonist (Ϯ)-BayK 8644 and the antagonist (Ϯ)isradipine were obvious. The addition of 10 M (Ϯ)-BayK8644 resulted in a 3.7 Ϯ 0.3-fold (n ϭ 5) stimulation of peak I Ba , measured at test potentials of 10 mV negative to the peak potential of the current-voltage relations (Fig. 4A). This amount of peak I Ba increase is significantly (p Ͻ 0.01) higher than that of ␣ 1A-DHPi -mediated I Ba (Fig. 4A) and corresponds to the extent expected for recombinant L-type channels expressed in Xenopus oocytes (18). Other classical features of BayK 8644 modulation were also restored in ␣ 1A-DHP . We observed a characteristic slowing in the activation kinetics, determined as a 62 Ϯ 28-ms (n ϭ 3) difference in the time to peak I Ba with and without drug at test potentials close to the midpoint voltage of the current activation curve. (Ϯ)-BayK 8644 shifted the acti-vation curve by 7.1 Ϯ 1.8 mV (n ϭ 3) in the hyperpolarized direction and induced a moderate but detectable slowing of deactivation (Fig. 3B, lower panel). The stimulatory effect of (Ϯ)-BayK 8644 was concentration-dependent (Fig. 4A). A complete concentration-response relationship could not be obtained due to the limited solubility of (Ϯ)-BayK 8644 in the recording solution. Nevertheless, the data obtained for ␣ 1A-DHPi and ␣ 1A-DHP (Fig. 4A) indicate that Met 1188 critically contributes to agonist modulation by (Ϯ)-BayK 8644. Despite a 1.6 Ϯ 0.1-fold (n ϭ 7) increase of peak I Ba at 10 M, (Ϯ)-BayK 8644 failed to appreciably shift the activation curve and slowed neither the activation kinetics nor the deactivation kinetics of ␣ 1A-DHPi (Fig. 1). Met 1188 therefore is likely to at least contribute to the ability of L-type calcium channels to display these features of agonist modulation, but this hypothesis remains to be confirmed by testing for the functional consequences of a respective Met to Val or Met to Ala mutation in ␣ 1C or ␣ 1S .
To investigate the effect of the Val to Met conversion on DHP antagonist sensitivity, we assessed the concentration-dependent inhibition of ␣ 1A-DHP by (Ϯ)-isradipine in comparison with ␣ 1A-DHPi (Fig. 4B). The apparent IC 50 values of ϳ10 M for ␣ 1A-DHPi and 67 nM for ␣ 1A-DHP indicate that Met 1188 exhibits also a substantial impact on DHP antagonist sensitivity. BayK 8644 -Fig. 5 illustrates the antagonist and agonist effects on ␣ 1A-DHP that are typical for L-type calcium channel modulation. Stereoselectivity represents a core requirement for the specificity of drug-receptor interactions and was unequivocally present in ␣ 1A-DHP . As shown in Fig. 5A, (ϩ)-isradipine displayed a clearly higher potency than (Ϫ)-isradipine in blocking I Ba of ␣ 1A-DHP . In addition, block of ␣ 1A-DHP by (Ϯ)-isradipine was voltage-dependent. DHP antagonists preferentially bind to the inactivated state of L-type channels, exhibiting a higher affinity at depolarized holding potentials (10), which is observed as an antagonist-induced shift of the steady-state inactivation curve to more negative voltages. Under control conditions, ␣ 1A-DHP exhibited steady-state inactivation with a V hϱ 1 ⁄2 of Ϫ49.5 Ϯ 0.9 mV (n ϭ 3), indistinguishable (p Ͼ 0.01) from the Ϫ52.4 Ϯ 3.4 mV (n ϭ 3) obtained for ␣ 1A-DHPi . In the presence of 0.1 M (Ϯ)-isradipine, the V hϱ 1 ⁄2 of ␣ 1A-DHP was shifted 10.0 Ϯ 1.8 mV (n ϭ 3) in the hyperpolarized direction (Fig. 5B), clearly demonstrating a state dependence of the antagonist effect.

Features of the Modulation of ␣ 1A-DHP by (Ϯ)-Isradipine and (Ϯ)-
Previous studies have demonstrated that DHP agonist modulation of L-type calcium channels is voltage-dependent (32)(33)(34). As shown in Fig. 5C, this feature was observed for the modulation of ␣ 1A-DHP by (Ϯ)-BayK 8644. At a pulse frequency of 0.1 Hz and a holding potential of Ϫ60 mV, the extent of peak I Ba stimulation by 10 M (Ϯ)-BayK 8644 was significantly (p Ͻ 0.05) reduced to 1.5 Ϯ 0.1-fold (n ϭ 3) (Fig. 5C, lower panel) when compared with the 3.9 Ϯ 0.5-fold (n ϭ 5) stimulation at a holding potential of Ϫ80 mV (Fig. 5C, upper panel). These results demonstrate that the P/Q-type channel mutant ␣ 1A-DHP exhibits features of DHP agonist and antagonist modulation that are usually observed for wild-type L-type channels.

Transfer of DHP Sensitivity in a Gain-of-function Ap-
proach-We demonstrate that the high affinity DHP interaction domain of L-type calcium channels can be transposed to the DHP-insensitive ␣ 1A subunit by simultaneous conversion of as few as nine amino acid residues to their L-type counterparts. The involvement of eight of these nine L-type residues in DHP interaction has previously been shown (19 -21). The importance of an additional L-type methionine residue (Met 1188 ; ␣ 1C-a numbering) to fully support DHP sensitivity represents a new finding presented in this study. The role of Met 1188 was uncovered by the differences in DHP agonist and antagonist sensitivity of the chimeric calcium channel ␣ 1 subunit AL12m, that was constructed by merging sequence stretches from the DHP-insensitive ␣ 1A subunit (22) and the ␣ 1M subunit, which had been cloned from M. domestica body wall muscle (24). AL12m, in addition to having provided an opportunity to identify the importance of Met 1188 for DHP interaction, enabled us to characterize for the first time the DHP sensitivity of an ancestral L-type ␣ 1 subunit (Fig. 2). The validity of a gain-of-function approach in the examination of the structural requirements for calcium channel modulation by DHPs was demonstrated by the P/Q-type calcium channel-derived mutant ␣ 1A subunit ␣ 1A-DHP . The simultaneous mutations Y1393T, M1397Q, F1504I, F1507I, V1512M, I1804Y, F1805M, M1811I, and L1812I (␣ 1A numbering) generated full sensitivity for the DHP agonists and antagonists (Ϯ)-BayK 8644 and (Ϯ)-isradipine, respectively (Fig. 3), the optical enantiomers of the DHP-(202-791), and even the structurally unrelated benzoyl pyrrole FPL 64176 (Fig. 6). It is important to point out that in addition to these residues a tyrosine (Tyr 1503 ; ␣ 1A numbering) and possibly other residues that are conserved between ␣ 1A and L-type calcium channel ␣ 1 subunits may also participate in the formation of this binding domain (20). ␣ 1A-DHP closely resembled native L-type channels with respect to DHP modulation. The typical hallmarks of L-type channel modulation, such as stereoselectivity and a shift of the steady-state inactivation curve to more negative voltages by the antagonist as well as the voltage dependence of agonist effects (32)(33)(34), were exquisitely preserved in ␣ 1A-DHP (Fig. 5). Half-maximal inhibition of peak I Ba by the DHP antagonist (Ϯ)-isradipine (Fig. 4B) occurred with an even higher apparent potency than that reported for recombinant L-type channels expressed in Xenopus oocytes (18). Since DHP antagonists bind with high affinity to the inactivated state of L-type channels (10) , and the non-DHP agonist FPL 64176. I Ba were elicited by depolarizations to potentials corresponding to the peak (antagonist) or 10 mV negative to the peak (agonist) of the currentvoltage relations. Pulse duration was 350 ms, and the holding potential was Ϫ80 mV. Capacitative transients were truncated for presentation. could be mediated by its steady-state inactivation properties. Indeed, a small fraction of inactivated channels was detected at a test potential of Ϫ80 mV (Fig. 5B) used in our experiments. However, comparable availability of ␣ 1A-DHPi and ␣ 1A-DHP , together with the ϳ150-fold difference in apparent potency of (Ϯ)-isradipine block between these mutants, favors a structurebased interpretation of the pronounced antagonist sensitivity obtained for ␣ 1A-DHP . Summarizing these findings, ␣ 1A-DHP represents an excellent example for the transfer of sensitivity to nonpeptide modulators from one voltage-gated ion channel to another, accomplished by the conversion of a minimal number of amino acid residues.
Implications for Channel Structure-The L-type residues that successfully transferred DHP sensitivity are located within transmembrane segments IIIS5, IIIS6, and IVS6 (Fig.  7). These regions appear to be particular hot spots of voltagedependent calcium channels with respect to drug interaction, since they also contain the molecular determinants for phenylalkylamine (35,36) and benz(othi)azepine interaction (26,37). DHPs, phenylalkylamines, and benz(othi)azepines even share Tyr 1490 and Ile 1497 (␣ 1C-a numbering) in transmembrane segment IVS6 as common interaction residues. These results are consistent with findings from in vitro binding studies employing fluorescent calcium channel ligands (38) and photoaffinity labeling (37), which demonstrated that the DHP and benz(othi)azepine binding domains are localized in close proximity to each other on the L-type ␣ 1 subunit. At least some of the residues that interact with these drugs seem to face the channel pore. This has previously been demonstrated for Tyr 1490 by an increase of N-methyl-D-glucamine conductivity following a Tyr to Ala conversion (35). To bridge the DHP molecule, the position of the interaction residues must be within a distance of approximately 13-15 Å (assuming a max-imal drug diameter of about 9 Å and bond lengths of 2-3 Å). Accordingly, transmembrane segments IIIS5, IIIS6, and IVS6 must lie in close vicinity to each other in the folded ␣ 1 subunit, with the DHP binding domain orientated toward the channel pore.
Transmembrane segments of voltage-gated ion channels are predicted to display an ␣-helical structure (39,40). Based on this model, all of the identified L-type residues interacting with DHPs align to the same side of the respective ␣-helices, with the exception of Met 1188 . This could indicate a more indirect effect of Met 1188 , mediated by changes in electrophysiological properties that result in an increase of DHP sensitivity. However, biophysical parameters that could affect DHP sensitivity, such as steady-state inactivation as well as I Ba activation, were not appreciably altered among the mutant channels ␣ 1A-DHPi and ␣ 1A-DHP . We therefore assume that a direct contribution of binding energy by Met 1188 is responsible for its importance in DHP interaction. Since this residue is located on the opposite side of Tyr 1179 and Ile 1180 (␣ 1C-a numbering) in a putative ␣-helix, this can only be accomplished if segment IIIS6 protrudes deeply into the pore. Alternatively, and similar to the S6 segment of Shaker potassium channels (40), portions of the cytoplasmic half of IIIS6 may be tilted or deviate from a straight ␣-helix. A specific example for this hypothesis is a proline (Pro 1508 ; ␣ 1A numbering), that is conserved in non-Ltype ␣ 1 subunits and therefore present in the IIIS6 segment of ␣ 1A-DHP . This residue separates Met 1188 from the Tyr 1179 -Ile 1180 -Ile 1183 DHP interaction motif and replaces an alanine (Ala 1184 ; ␣ 1C-a numbering) that is conserved throughout L-type ␣ 1 subunits. In analogy to the model of potassium channel S6 segments (40), this proline residue could produce a tilt in the putative ␣-helix of transmembrane segment IIIS6. As a result Met 1188 might achieve a more favorable orientation for drug interaction in ␣ 1A-DHP than in native L-type channels, which could in part explain the remarkably high DHP sensitivity of this mutant. The consequences of a Pro to Ala conversion in transmembrane segment IIIS6 of ␣ 1A-DHP on functional DHP modulation and radioligand binding will challenge this hypothesis.
In conclusion, the gain-of-function mutant ␣ 1A-DHP represents a valuable tool to investigate the structure-activity relationship between DHPs and the molecular determinants of their interaction domain. Expression in mammalian cells and radioligand binding studies as well as electrophysiological analysis of the resulting mutants will provide a suitable assay for an even more refined study. Further characterization of the structural determinants of channel gating is under way and, together with our findings, will eventually unravel the molecular mechanism of channel modulation by DHPs. Elucidation of the structure-activity relationship between ␣ 1A-DHP and DHPs will provide useful molecular information that may be exploited to design and develop compounds that selectively interact with non-L-type calcium channels. Such drugs could be of therapeutic value for the treatment of mental disorders, stroke, and pain (41). Most recently, certain forms of migraine and ataxia have been linked to mutations in the ␣ 1A subunit gene (42,43), which are expected to result in altered channel activity. Selective drugs, therefore, may also be effective in the treatment of diseases associated with changes in P/Q-type channel function.