Introduction

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L-type voltage-gated Ca²⁺ channels play a critical role in the initiation of muscle contraction in the cardiovascular system and also contribute to the timing of the cardiac action potential (Bers and Perez-Reyes, 1999). Therefore, drugs that inhibit Ca²⁺ flux through L-type Ca²⁺ channels are used to treat hypertension, angina pectoris, and some cardiac arrhythmias. Three major classes of Ca²⁺ channel blockers are currently in clinical use: dihydropyridines (DHP), phenylalkylamines (PA), and benzothiazepines (BZP). Ca²⁺ channels are composed of a large, pore-forming ₁ subunit consisting of four homologous domains (I-IV), each containing six transmembrane segments (S1-S6), in a complex with auxiliary , , and ₂ subunits (Takahashi et al., 1987; Tanabe et al., 1987; Catterall, 1995). L-type Ca²⁺ channels in the heart and vascular smooth muscle contain _1c subunits, which are highly sensitive to Ca²⁺ channel blocking drugs, whereas the drug-insensitive N-type and P/Q-type Ca²⁺ channels contain _1B and _1A subunits, respectively (reviewed in Hofmann et al., 1999).

Drugs from all three of these classes bind to L-type Ca²⁺ channels, and their binding sites are closely linked but not identical (Gould et al., 1983). Biochemical experiments utilizing photoreactive derivatives of DHPs and PAs indicated interaction of DHPs with domains IIIS6 and IVS6 (Nakayama et al., 1991; Striessnig et al., 1991), and PAs with domain IVS6 of the ₁ subunit (Striessnig et al., 1990). Subsequent experiments using site-directed mutagenesis and electrophysiological recordings of recombinant channels expressed in various cell types have led to a detailed picture of the amino acid residues required for modulation of L-type Ca²⁺ channels by DHPs and PAs (Hockerman et al., 1997b; Mitterdorfer et al., 1998; Hofmann et al., 1999).

The site of action for BZPs has been less extensively characterized. Diltiazem, a clinically relevant member of the BZP class of drugs, has several unique properties that suggest that its binding site is distinct from that of DHPs or PAs. Diltiazem stimulates the binding of DHPs but inhibits the binding of PAs to L-type Ca²⁺ channels (DePover et al., 1982; Ferry and Glossmann, 1982) suggesting that there are elements of the DHP binding site that are distinct from the BZP binding site. In addition, block of L-type Ca²⁺ channels by diltiazem is frequency-dependent, similar to but less pronounced than the frequency-dependent block by PAs (Lee and Tsien, 1983). This suggests that both diltiazem and PAs bind preferentially to the open and/or inactivated states of the channel, and may therefore share some common binding determinants within the channel. Finally, diltiazem shows lower selectivity for L-type versus non-L-type Ca²⁺ channels than DHPs or PAs (Diochot et al., 1995; Ishibashi et al., 1995). This suggests that many of the binding determinants for diltiazem are identical across Ca²⁺ channel types, or that differences at these positions are conservative enough to allow diltiazem binding.

Biochemical experiments using a photoreactive BZP reported derivatization of transmembrane segments IIIS6 and IVS6 of L-type Ca²⁺ channels (Kraus et al., 1996). Subsequent studies using chimeric Ca²⁺ channels with IIIS6 and IVS6 segments from _1C spliced into an _1A background found that many of the same amino acid residues critical for high affinity PA block (Hockerman et al., 1995, 1997a) were also involved in diltiazem block (Hering et al., 1996; Kraus et al., 1998). To date, no studies have systematically examined the role of each amino acid residue in the IIIS6 and IVS6 segments. Therefore, we have constructed single amino acid mutations to alanine along the entire IIIS6 and IVS6 segments of _1C and have analyzed each mutant for sensitivity to block by diltiazem. In addition, we have measured the sensitivity to diltiazem block of both the wild-type _1A (P/Q-type) channel and an _1A mutant (_1A/DHPS) that contains the DHP binding site. To assess the contribution to diltiazem block of amino acid residues in transmembrane domain IIIS5 known to be critical for DHP block (Grabner et al., 1996; He et al., 1997), we have constructed a double amino-acid mutation in IIIS5 (_1C/DHPI) and analyzed it for sensitivity to both the DHP PN200-110 and diltiazem. Our results reveal several amino acid residues that contribute to block by diltiazem but not by DHPs and/or PAs; conversely, several key amino acid residues contribute to block by DHPs and/or PAs but do not contribute to block by diltiazem.

	Experimental Procedures

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Construction of Mutants. Single amino acid mutations in domains IIIS6 and IVS6 of the _1C subunit (Snutch et al., 1991) were constructed using oligonucleotide-directed mutagenesis, as described previously (Kunkel, 1985). The IIIS6 mutations were inserted into full-length subunit constructs in the expression vector Zem 229 (Dr. Eileen Mulvihill, University of Washington, Seattle, WA) using the 1.5-kilobase SpeI/DraIII fragment and the 272 bp DraIII/DraIII fragment in a three-way ligation. Construction of the _1A/DHPS mutant was described previously (Hockerman et al., 1997c). The _1C/DHPI mutant contains two mutations in domain IIIS5 (T1039 to Y and Q1043 to M) and was constructed using the splice overlap extension method (Horton et al., 1989). The mutant 600-bp DNA fragment was cut with SpeI and BglII and inserted into the full-length subunit construct in the expression vector Zem 229 using the 900-bp BglII//DraIII fragment and the 272-bp DraIII/DraIII fragment in a four-way ligation. All mutations were confirmed by cDNA sequencing.

Cell Culture. tsA201 cells, a subclone of the human embryonic kidney cell line 293 that expresses simian virus 40 T antigen (a gift of Dr. Robert Dubridge, Cell Genesis, Foster City, CA) were maintained in monolayer culture in Dulbecco's modified Eagle's medium/Ham's F-12 medium (Life Technologies, Inc., Gaithersburg, MD), supplemented with 10% fetal bovine serum (Hyclone, Logan, UT), and incubated at 37°C in 10% CO₂.

Expression. Wild-type and mutant _1C channel subunits (Snutch et al., 1991) were expressed with _1B (Pragnell et al., 1991) and ₂ channel (Ellis et al., 1988) subunits and either CD8 antigen (EBO-pCD-Leu2, American Type Culture Collection, Manassas, VA) or enhanced green fluorescent protein (CLONTECH, Palo Alto, CA) in tsA201 cells by transfection with either Ca²⁺ phosphate (Margolskee et al., 1993) or the transfection reagent GenePorter (Gene Therapy Systems, San Diego, CA). Transfectants were selected by labeling with anti-CD8 antibodies conjugated to polystyrene beads (Dynal, Oslo, Norway) or by fluorescence at 510 nm with excitation at 480 nm (enhanced green fluorescent protein transfected cells).

Electrophysiology. Ba²⁺ currents through Ca²⁺ channels were measured using the whole-cell, patch-clamp configuration. Pipettes were pulled from VWR micropipettes (VWR, West Chester, PA) and fire polished to produce an inner tip diameter of 4 to 6 µm. Currents were recorded using an Axon Instruments Axopatch 200B amplifier and filtered at 1 or 2 kHz (six-pole Bessel filter, 3 dB). Voltage pulses were applied and data were acquired using pClamp6 software (Axon Instruments, Foster City, CA). Voltage-dependent currents have been corrected for leak using an online P/4 subtraction paradigm. (+)-cis-Diltiazem (RBI, Natick, MA) dissolved in bath saline was applied to cells under voltage clamp using a fast perfusion system with constant exchange of the bath solution. PN200-110 was added to the bath without background perfusion as a 3× stock. The bath saline contained 150 mM Tris, 10 mM BaCl₂, and 4 mM MgCl₂. The intracellular solution contained 130 mM N-methyl-D-glucamine, 10 mM EGTA, 60 mM HEPES, 2 mM MgATP, 1 mM MgCl₂. The pH of both solutions was adjusted to 7.3 with methanesulfonic acid. All experiments were done at room temperature (20-23°C).

	Results

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Block of Ca²⁺ Channels Containing Wild-Type _1C and _1A Subunits by (+)-cis-Diltiazem. The _1C subunit (Snutch et al., 1991) was expressed in tsA201 cells along with the _1B (Pragnell et al., 1991) and ₂ (Ellis et al., 1988) subunits. Ba²⁺ currents through the resulting L-type channels were blocked by (+)-cis-diltiazem with an IC₅₀ value of 33.3 ± 4.6 µM (Fig. 1A, F). For L-type channels, a holding potential of 60 mV with a 100-ms test pulse to +10 mV at a frequency of 0.05 Hz was used. Under these conditions, block developed rapidly, and reached equilibrium within 200 s (Fig. 1B). Little (<10%) frequency-dependent block accumulated using this low stimulation frequency. These conditions were used for the assay of diltiazem sensitivity of all the _1C mutants.

View larger version (24K): [in this window] [in a new window]   Fig. 1.   Diltiazem block of wild-type 1C, 1A, 1C/DHPI, and 1A/DHPS. A, representative Ba2+ current records from a tsA201 cell expressing wild-type 1C in the absence (control) or presence of the indicated concentrations of diltiazem. B, time course of diltiazem block of 1C Ba2+ current expressed in a tsA201 cell. (+)-cis-diltiazem was applied at the pulse number and concentrations indicated. Pulses were to +10 mV from a holding potential of 60 mV every 20 s. C, Representative current records from a tsA 201 cell expressing wild-type 1A in the absence (control) or presence of the indicated concentrations of diltiazem. D, representative current records from a tsA201 cell expressing the mutant 1A/DHPS in the absence (control) or presence of the indicated concentrations of diltiazem. E, time course of block of wild-type 1C () and 1C/DHPI () by the DHP PN200-110 (10 µM). F, summary of the IC50 values of wild-type 1C, wild-type 1A, 1A/DHPS, and 1C/DHPI for (+)-cis-diltiazem. The IC50 values (shown ± S.E.) were determined as described in the legend to Fig. 2. The asterisks denote that the IC50 values for 1C, 1A/DHPS, and 1C/DHPI are significantly different from the IC50 value for 1A (Student's t test, * P < .05, ** P < .01). Note that 1C/DHPI has the same sensitivity to diltiazem as wild type 1C even though it is virtually insensitive to PN200-110.

Block of Mutant Ca²⁺ Channels Containing _1A/DHPS and _1C/DHPI by (+)-cis-Diltiazem. We hypothesized that the difference in sensitivity to diltiazem block between the wild-type _1C and _1A channels might be caused by changes in relatively few amino acid residues. As an initial experiment, we tested the diltiazem affinity of a mutant _1A channel in which nine amino acids from _1C had been substituted. These substitutions had been shown previously to confer nearly full sensitivity to DHPs (Hockerman et al., 1997c). We expressed this mutant, _1A/DHPS, along with the _1B and ₂ subunits in tsA201 cells and applied increasing concentrations of (+)-cis-diltiazem to cells under voltage clamp using a holding potential of 120 mV and 100-ms test pulses to 0 mV at a frequency of 0.033 Hz. The strongly negative holding potential was necessary to compensate for the very negative voltage dependence of inactivation of the _1A/DHPS mutant (Hockerman et al., 1997c). Diltiazem blocked Ba²⁺ currents through _1A/DHPS with an IC₅₀ value of 35.5 ± 13.6 µM, a concentration virtually identical with that of wild-type _1C (Fig. 1, D and F).

Effects of Single Amino Acid Alanine Substitution Mutants in Transmembrane Segment IIIS6 on Block by (+)-cis-Diltiazem. Because photoaffinity labeling studies suggest that diltiazem interacts with both transmembrane domains IIIS6 and IVS6 (Kraus et al., 1996), we screened a battery of single amino acid mutations to alanine spanning both the IIIS6 and IVS6 transmembrane domains of _1C for concentration-dependent block by diltiazem. At positions where the native amino acid is alanine, we substituted the corresponding amino acid in non-L-type channels (A1157P, A1467S). We also made more conservative changes at tyrosines 1152 and 1463, mutating them to phenylalanine. We expressed the _1C mutant channels in tsA201 cells along with the _1B and ₂ subunits and added increasing concentrations of (+)-cis-diltiazem to cells under voltage clamp using a holding potential of 60 mV and test pulses to +10 mV for 100 ms at 0.05 Hz. Of the single amino acid mutations tested, only A1157P did not express current. The functional properties of the mutant channels that were expressed at low level have been described previously (Hockerman et al., 1997a). Of the 21 single amino acid mutants in segment IIIS6 that we screened, eight had no significant change in diltiazem affinity, and six had <2-fold change in the IC₅₀ value for diltiazem (Fig. 2). In contrast, mutants I1150A, I1153A, I1156A, and M1160A in the central portion of the segment had substantially reduced sensitivity to diltiazem block compared with wild-type _1C (I1150A, IC₅₀ = 97.1 ± 20.6 µM; I1153A, IC₅₀ = 93.1 ± 24.4 µM; I1156A, IC₅₀ = 83.6 ± 10.9 µM; M1160A, IC₅₀ = 107.9 ± 7.7 µM). In addition, mutation of two amino acid residues near the intracellular end of this transmembrane segment, F1164A and V1165A, also resulted in substantially decreased potency of diltiazem block of Ba²⁺ current (F1164A, IC₅₀ = 90.8 ± 9.4 µM; V1165A, IC₅₀ = 131 ± 40 µM).

View larger version (29K): [in this window] [in a new window]   Fig. 2.   Diltiazem block of wild-type 1C channels and 1C channels with single-amino-acid mutations in IIIS6. A, L-type Ba2+ currents were recorded from wild-type and mutant 1C L-type channels as described under Experimental Procedures. Representative traces are shown in the absence (control) or presence of the indicated concentrations of (+)-cis-diltiazem for wild-type amino acids (left), the mutant I1150A (center), and the mutant M1160A (right). B, dose-response relationships for the indicated wild type and mutant channels. In each case, the averaged, normalized current amplitudes at 5, 10, 50, 100, and 500 µM diltiazem (symbols ± S.E.) were plotted against the corresponding drug concentration, and the IC50 value was determined by fitting the averaged relative current values at each diltiazem concentration to the equation, relative current = 1 {1/[1+(IC50/[diltiazem])]} (smooth lines). C, the IC50 values of the indicated mutations in IIIS6 for diltiazem are shown ± S.E. Asterisks denote that the IC50 value for diltiazem of the indicated mutant channel is significantly different from that of wild-type 1C (Student's t test: * P < .05, ** P < .01, *** P < .001).

Effects of Block of Single-Amino-Acid Alanine Substitution Mutants in Transmembrane Segment IVS6 by (+)-cis-Diltiazem. We also screened single-amino-acid mutations across transmembrane IVS6 for concentration dependence of diltiazem block as described above. The functional properties of these mutant Ca²⁺ channels have been described previously (Hockerman et al., 1995). Of the 13 amino acids in IVS6 that were mutated, only mutant channels I1460A, Y1463F, and M1464A showed decreased sensitivity to diltiazem (I1460A, IC₅₀ = 69.5 ± 7.5 µM; Y1463F, IC₅₀ = 114.4 ± 19 µM; M1464A, IC₅₀ = 91.8 ± 8.9 µM; Fig. 3).

View larger version (25K): [in this window] [in a new window]   Fig. 3.   Diltiazem block of wild-type 1C channels and 1C channels with single-amino-acid mutations in IVS6. A, L-type Ba2+ currents were recorded from wild-type and mutant 1C L-type channels as described under Experimental Procedures. A, representative traces are shown in the absence (control) or presence of the indicated concentrations of (+)-cis-diltiazem for wild-type (left), the mutant I1460A (center), and the mutant M1464A (right). B, dose-response relationships are shown for the indicated wild-type and mutant channels and are as described in the legend to Fig. 2. C, the IC50 values of the indicated mutations in IVS6 for diltiazem are shown ± S.E. and were determined as described in the legend to Fig. 2. An asterisk denotes that the IC50 value for diltiazem of the indicated mutant channel is significantly different from that of wild-type 1C (Student's t test, * P < .05, *** P < .001).

	Discussion

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Distinct but Overlapping Binding Sites for BZPs and DHPs on _1C. Using the _1A/DHPS channel, we have shown that insertion of a DHP binding site into a channel that is normally insensitive to DHP increases the sensitivity to block by diltiazem by 5-fold, making the IC₅₀ value of diltiazem for this mutant _1A channel virtually identical with the wild-type _1C (Fig. 1, D and F). Because _1A/DHPS contains amino acid substitutions in three transmembrane segments, IIIS5, IIIS6, and IVS6, we assessed the contribution of amino acid residues in these three regions to diltiazem block. Mutation of both T1039 and Q1043 in IIIS5 of _1C (mutant _1C/DHPI), removes DHP block (Mitterdorfer et al., 1996; He et al., 1997) (Fig. 1E) and virtually eliminates DHP binding (He et al., 1997), but does not affect block by diltiazem (Fig. 1F). Therefore, T1039 and Q1043 in IIIS5 are clearly involved in DHP block, but not diltiazem block, of _1C. Similarly, Y1152, F1158, F1159, and M1161 in transmembrane segment IIIS6 as well as I1470, I1471, and N1472 in segment IVS6 are required for DHP binding and block but not for BZP block (Fig. 4). Thus, nine amino acid residues in these three transmembrane segments are involved in the DHP receptor site but not the BZP receptor site.

View larger version (47K): [in this window] [in a new window]   Fig. 4.   Amino acid residues required for binding of calcium antagonist drugs. Top, amino acid sequences of transmembrane segments IIIS5, IIIS6, and IVS6 of 1A, 1C, and 1A/DHPS. Amino acid residues in bold type are required for high-affinity BZP binding. Amino acid residues in bold italic type are required for high-affinity BZP binding, but not included in 1A/DHPS. Bottom, transmembrane -helical segments, with amino acid residues required for high-affinity binding of DHPs, BZPs, and PAs indicated as .

Similar but Nonidentical Binding Sites on _1C for Diltiazem and Phenylalkylamines. Diltiazem and PAs block L-type Ca²⁺ channels in a similar manner. They are less potent than DHPs and, unlike DHPs, they exhibit striking frequency-dependent block (Lee and Tsien, 1983). Previous studies have implicated transmembrane segments IIIS6 and IVS6 in block of L-type Ca²⁺ channels by the high-affinity PA ()-D888 (Hockerman et al., 1995, 1997a; Doring et al., 1996; Hering et al., 1997). Of the seven amino acid residues that may interact directly with BZPs, only I1153 in segment IIIS6 and Y1463 in segment IVS6 are also important for block by PAs (Fig. 4). I1150, I1156, and M1160 in segment IIIS6 as well as I1460 and M1464 in segment IVS6 are required for BZP binding but not PA binding (Fig. 4). Interestingly, F1164 and V1165, which are not implicated in DHP block, have been proposed as key determinants in the frequency dependence of both PA and BZP block (Hering et al., 1997; Kraus et al., 1998). Both F1164 and V1165 are conserved between _1C and _1A, and are therefore present in the _1A/DHPS mutant, where they most likely contribute substantially to diltiazem action. As noted above, it is likely that these residues are indirect allosteric effectors of state-dependent binding of PA and BZPs.

Amino Acid Residues Involved in Diltiazem Block of _1C Are Conservatively Substituted in _1A. A surprising finding of this study is the relatively small difference between the IC₅₀ values for diltiazem block of wild-type _1C and _1A. Diltiazem has been considered a relatively specific blocker of L-type Ca²⁺ channels, although block of other channel types at higher concentrations has been noted (Diochot et al., 1995; Ishibashi et al., 1995). Our findings suggest that the selectivity of diltiazem for L-type channels over P/Q-type channels is less than an order of magnitude. Most of the determinants of diltiazem block reported here are not conserved between _1C and _1A. How then, can the P/Q-type channel retain its relatively high degree of sensitivity to diltiazem?