Motif III S5 of L-type Calcium Channels Is Involved in the Dihydropyridine Binding Site
A COMBINED RADIOLIGAND BINDING AND ELECTROPHYSIOLOGICAL STUDY*

(Received for publication, December 2, 1996)

Ming He , Ilona Bodi , Gabor Mikala and Arnold Schwartz Dagger

From the Institute of Molecular Pharmacology and Biophysics, University of Cincinnati, Cincinnati, Ohio 45267-0828

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
Acknowledgments
Note Added in Proof
REFERENCES


ABSTRACT

The alpha 1 subunit of L-type voltage-dependent Ca2+ channels (alpha 1C) has been shown to harbor high affinity binding sites for the Ca2+ channel dihydropyridine (DHP) modulators. It has been suggested by a number of investigators that the binding site may be composed of III S6 and IV S6. Evidence with chimeric channels indicated the possible involvement of III S5 in DHP binding. Site-directed mutations were introduced in motif III S5 region of the alpha 1C, changing the amino acids to their counterparts in the DHP-insensitive alpha 1A channel. The mutant channels were expressed both in HEK 293 cells and in Xenopus oocytes. Equilibrium binding and electrophysiological studies showed that the Thr1006 to Tyr substitution produced a mutant channel with at least 1000-fold decreased affinity in [3H](+)isopropyl-4-(2,1,3-benzoxadiazol-4-yl)-1,4-dihydro-(2,6-dimethyl-5-methoxycarbonyl)pyridine-3-carboxylate (PN200-110, isradipine) binding and in sensitivity of R(-)-4(2,1,3-benzoxadiazol-4-yl)-1,4-dihydro-2,6-dimethyl-5-nitro-3-pyridincarboxylic acid isopropylester (R202-791) in terms of inhibition of current through the L-type voltage-dependent Ca2+ channels. Replacing Gln1010 with Met resulted in more than a 10-fold decrease in binding affinity for [3H](+)PN200-110 and in the potency of channel modulation by S202-791. Four additional mutations in this region also lead to a slight but statistically significant increase of KD values for [3H](+)PN200-110 binding. The binding and electrophysiological results show that certain residues of the transmembrane segment III S5 are important in contributing to the DHP binding "pocket" and are critical for DHP binding and for its calcium channel effect.


INTRODUCTION

Voltage-dependent Ca2+ channels (VDCC)1 are divided into two subfamilies, the DHP-sensitive L-VDCC subfamily and the DHP-insensitive VDCC subfamily (1). Electrophysiological studies using charged DHPs pointed out that the binding site is accessible exclusively from the outside membrane (2, 3). Previous experimental evidence using photoaffinity labeling and peptide antibody mapping suggested that S6 of motif III and IV plus part of their adjacent extracellular loops (4, 5) as well as a putative "E-F hand" flanking region (6) are involved in forming the binding "pocket". These results were in part supported by molecular studies. Because there is a considerable sequence homology between the residues of the DHP-sensitive VDCC alpha 1 and the DHP-insensitive VDCC alpha 1 subunits, construction of chimeric channels proved to be a logical method to identify regions involved in the drug binding sites. It was shown that part of IV S6 and the linker of IV S5/S6 were critical for the DHP agonist effect (7). In order to further define the regions responsible for DHP binding and pharmacological action, single amino acids in III S6 and IV S6 were mutated, and radioligand binding demonstrated that these mutants had reduced binding affinity to DHPs (8, 9). Moreover, electrophysiological studies suggested that the amino acids in IV S6 may contribute differentially to the effect of DHP agonist and antagonist (7, 9, 10). The importance of these regions was further confirmed by introducing III S5-III S6 and IV S6 along with part of IV S5/S6 linker of alpha 1C channel into the alpha 1A channel (10). This alteration was sufficient to confer sensitivity to both DHP agonist and antagonist to the alpha 1A channel.

At the same time, we found that by replacing the III S5-S6 of the alpha 1C with that of the corresponding region of alpha 1A, the resulting Ca2+ channels lost sensitivity to both DHP agonist (-)BayK 8644 and antagonist (+)BayK8644 (11). If III S6 was replaced by the corresponding region of alpha 1A, the chimeric channel became insensitive to (+)BayK8644, and the EC50 of (-)BayK8644, the agonist, was increased 10-fold compared with the wild type channel (11). We speculated that even though III S5 was not photoaffinity labeled, it may form an important part of interaction sites for DHPs distal from the photoactive group. We feel that these mutations in III S5 cause alterations in the binding of DHPs to the channel, which in turn decreases the response of the channels to these drugs. Because DHP binding is done on membranes that are electrically neutral, and electrophysiological effects are voltage- and state-dependent, it is difficult to interpret whether mutations cause only an alteration of the binding pocket and/or if changes in other functional properties of the channel occur. It is critical, therefore, to study the coupling between DHP binding and modulation of current. Thus, we mutated nonconserved amino acids of III S5 in the alpha 1C channel (12, 13) to those in the alpha 1A channels (14) and tested the mutants using both radioligand binding and electrophysiological studies. We found consistent results in terms of binding affinity and pharmacological sensitivity of these mutants to DHPs, strongly suggesting the importance of S5 in repeat III of the alpha 1 subunit of the L-VDCC.


EXPERIMENTAL PROCEDURES

Construction of alpha 1c cDNA Clones

The wild type and mutant rabbit heart L-VDCC alpha 1 subunit cDNAs were constructed in pBluescript II SK(-) in the T7 orientation for expression in the Xenopus oocyte system as described (7). Site-directed mutations were introduced by the PCR (Hoffman-LaRoche) in two steps. NsiI and PmlI restriction sites were used to insert the PCR products. Oligonucleotides carrying the PmlI site or having the desired mismatching bases served as forward primers, whereas oligonucleotides having the NsiI site served as the reverse primer. The cassette in the wild type alpha 1 subunit was replaced by the PCR products, and the existence of point mutations was verified by dideoxynucleotide sequencing (Amersham Corp.). For mammalian HEK 293 cell expression, mutant and wild type alpha 1 cDNAs were transferred from pBluescript II SK(-) into the mammalian expression vector pAGS-3 (15) at HindIII and NotI sites.

Transient Expression of Ca2+ Channels in HEK 293 Cells

HEK 293 cells were transfected with alpha 1, alpha 2/delta a (16), and human beta 3 (17) subunits at a 1:2:2 molar ratio by the calcium phosphate precipitation technique (18). Cells were harvested by scraping 48-72 h post transfection. Cells were washed twice with phosphate-buffered saline and disrupted in homogenizing buffer (10 mM Tris, pH 7.4, 2.0 µg/ml aprotinin, 0.5 mM phenylmethylsulfonyl fluoride). The membranes were pelleted by centrifugation at 36,000 rpm for 40 min and resuspended in storage buffer (50 mM Hepes-NaOH, 1.37 mM MgCl2, 1 mM EDTA, pH 7.4, 2 µg/ml aprotinin, 0.5 mM phenylmethylsulfonyl fluoride). Protein concentrations were measured using a BCA assay kit (Pierce).

Radioligand Binding

Saturation binding assays of [3H](+)PN200-110 (Amersham Corp.) were performed according to Varadi and co-workers (19) using 100 µg of protein for each sample in the binding buffer (100 mM Tris, 1.4 mM MgCl2, 2.4 mM CaCl2, pH 7.4) at 25 °C for 1 h. Nonspecific binding was determined using 1 µM unlabeled (±)PN200-110. Membranes were collected on glass fiber filters (FP-200 GF/C) and then washed with 50 mM Tris buffer (pH 7.4). Bound radioactivity was measured by liquid scintillation counting.

Xenopus Oocyte Expression

Expression of the wild type and mutant calcium channels in oocytes was done as described previously (7). In order to achieve maximal expression, the alpha 1c subunit cRNAs were coinjected with cRNAs specific for the alpha 2/delta a (16) and human beta 1b (20) subunits at a 1:1:1 molar ratio. Ca2+-activated Cl- channel contamination of Ba2+ currents was eliminated by microinjecting the oocytes with 50 nl of a solution containing 100 mM 1,2-bis(2-aminophenoxy)-ethane-N,N,N',N'-tetraacetate, 1 mM Tris, pH 7.4, 60 min preceding current recording. Whole cell currents were recorded with a two-electrode voltage-clamp amplifier (Axoclamp 2A). Voltage pulses were applied from a holding potential (HP) of -30 mV to test potentials between -30 mV and +40 mV in 10 mV increments. Whole cell leakage and capacitive currents were subtracted on line using the P/4 procedure. Currents were digitized at 1 kHz after being filtered at 1 kHz. The pClamp software (Axon Instruments, Burlingame, CA) was used for data acquisition and analysis. Only oocytes that showed less than 15% rundown over the first minutes were included in the analysis. The peak inward Ba2+ currents in the control solution and after applying each concentration of DHP compounds were plotted against DHP concentrations, and the curves were fitted to the following equation:
I<SUB><UP>Ba</UP><SUP>2<UP>+</UP></SUP></SUB>/I<SUB><UP>Ba</UP><SUP>2<UP>+</UP></SUP></SUB>(<UP>control</UP>)=1/(1+(C/C<SUB>50</SUB>)<SUP>h</SUP>) (Eq. 1)
where IBa2+ is Ba2+ current, C is the concentration of DHP compounds, C50 is the half-effective (inhibitory) concentration, and h is the Hill coefficient.

Chemicals

Stereoisomers of S202-791 were purchased from Biomol Research Laboratories Inc. (Plymouth Meeting, PA). [3H](+)PN200-110 was purchased from Amersham Corp. All other chemicals were from Sigma.

Statistical Analysis

The results were analyzed by the Radlig program version 4.0 (Biosoft) and Kaleidagraph software. The data are presented as the means ± S.E. The statistical analyses necessary were performed using Student's paired t test, with p values lower than 0.05 considered as indicator of significant difference.


RESULTS AND DISCUSSION

Our previous work investigated the contribution of motif IV to DHP effects by testing chimeric alpha 1 subunits engineered between the DHP-sensitive rabbit heart L-VDCC (Ca9) and the DHP-insensitive brain BI-2 (alpha 1A) subunits (7). We then further studied the contribution of other motifs to DHP effects. Our preliminary data (11) indicated that by replacing motif III S5-S6 of Ca9 (wild type) with that of the corresponding region of the alpha 1A, DHP sensitivity was lost.

A two-pronged experimental approach was launched. Following site-directed mutagenesis to change nonconserved amino acids in alpha 1C (Ca9) into the corresponding amino acids of alpha 1A subunit, each mutant was transiently coexpressed in HEK 293 cells with the accessory alpha 2/delta a and beta 3 subunits, and the "cardiac DHP receptor" was tested for DHP affinities using [3H](+)PN200-110 by equilibrium binding assays. In parallel, the same mutants were expressed in Xenopus oocytes, and the Ca2+ channel sensitivity to DHP agonists and antagonists was tested. [3H](+)PN200-110 binds to the membranes harboring wild type channel with a dissociation rate (KD) of 0.125 ± 0.019 nM. A hyperbolic saturation curve and a linear Scatchard plot indicate a single class of binding sites (Fig. 1B). We initiated our study by mutagenizing Thr1006, which starts the second putative alpha -helical turn of III S5. Mutation of Thr1006 to Tyr caused a dramatic decrease in the apparent affinity of the Ca2+ channel toward DHPs. At 25 nM [3H](+)PN200-110, only slight specific binding was detectable. At 75 nM of [3H](+)PN200-110, there was an increase in specific binding. These results indicate that the KD of T1006Y may be close to 100 nM. However, because specific binding can only be observed when high concentrations of ligand is used, it is difficult to make a quantitative analysis for the binding properties of DHPs to this mutant. We next mutated Gln1010, which may be adjacent to Thr1006 if one assumes an alpha -helical structure for III S5. Q1010M had a KD of 1.612 ± 0.395 nM, which is also significantly different from that of the wild type channel. These results suggest that Thr1006 and Gln1010 might be part of the tertiary structure forming the "DHP binding pocket."


Fig. 1. DHP binding characteristics of selected mutants in III S5 of the cardiac Ca2+ channel. A, upper, schematic representation of alpha 1C motif III of the voltage-dependent Ca2+ channel. Lower, alignment of III S5 amino acid sequences of alpha 1C and alpha 1A. The numbers refer to the alpha 1C sequence. B, the saturation binding curves of wild type, T1007M, and Q1010M to the DHP antagonist [3H]PN200-110. C, the KD values from the saturation curve fittings are presented by horizontal bars to the right. The names of the mutants are indicated to the left. The data are means ± S.E. A p value smaller than 0.05 is represented by an asterisk. Two asterisks represent a p value < 0.005. The open end of the horizontal bar of T1006Y represents an estimated value. D, Scatchard plots of the saturation binding data of wild type, T1007M, and Q1010M.
[View Larger Version of this Image (33K GIF file)]


In order to investigate other nonconserved residues of III S5, we then mutated Cys1015 and Gly1017 to their counterparts Val and Ala in the DHP-insensitive alpha 1A channel, because they may participate in disulfide bridges or turns, thereby contributing to the overall structure of the DHP binding domain. C1015V and G1017A mutants revealed a KD value for [3H](+)PN200-110 of 0.261 ± 0.016 nM and 0.180 ± 0.011 nM, respectively. This demonstrates that Cys1015 and Gly1017 are probably not important structural members for DHP binding. Because the residues Thr1006 and Gln1010 played an important role in DHP binding, we further mutated the adjacent amino acids Thr1007, Leu1009, and Met1012 to the corresponding amino acids in the alpha 1A channel. T1007M, L1009F, and M1012I mutant membranes had KD values indicating a 2-3-fold decrease in affinity. We speculate that these mutations may only slightly change the conformation of the DHP binding pocket.

In order to see whether the mutations that are associated with the changes described reflect corresponding changes in the channel action, T1006Y, T1007M, and Q1010M channels were expressed in Xenopus oocytes. Every mutant expressed functional voltage-dependent Ca2+ channels. All of the mutants exhibited a current activation threshold between -30 and -20 mV, and the peak current was reached at +10 to +20 mV test potential. The basic properties of current activation and inactivation were not affected by the mutations. The average of the peak inward current amplitude was 489.4 ± 39.3 (n = 48), 930.3 ± 248.1 (n = 13), 601.4 ± 64.3 (n = 22), and 390.2 ± 57.3 nA (n = 25) for the wild type, T1006Y, T1007M, and Q1010M channels, respectively.

The effects of the DHP agonist (+)S202-791 and antagonist (-)R202-791 were tested on T1006Y, T1007M, Q1010M, and the wild type. T1006Y completely lost the sensitivity to both agonist and antagonist (Fig. 2). After applying 10 µM agonist (+)S202-791 or antagonist (-)R202-791, we observed an approximately identical 23% decrease in the peak current that is likely due to a run-down during the experiment (Table I). T1007M showed a slight decrease of sensitivity to the agonist, but this difference was not statistically significant (Table I). The sensitivity to the antagonist of this mutant was slightly decreased compared with the wild type channel. After application of 1 µM and 10 µM (-)R202-791, the peak current was decreased by 40 and 78%, respectively (Table I), and it was associated with an accelerated inactivation similar to the wild type. Q1010M partially retained sensitivity to both agonist and antagonist. The peak current was increased by 1.65-fold after application of 10 µM (+)S202-791 and decreased by 37% after 10 µM (-)R202-791. However, these effects were significantly smaller than those seen with the wild type. The calculated EC50 values were 0.33, >10, 0.45, and 3.2 µM for the wild type, T1006Y, T1007M, and Q1010M, respectively. The calculated IC50 values were 0.13, 7.25, 0.66, and 3.82 µM for the above listed clones. In Fig. 3, the current-voltage relations of the Ba2+ current for the wild type and selected mutant channels are shown. Like the wild type channel, the T1007M responded to (+)S202-791 with both enhancement of current amplitude as well as by a shift of the current-voltage relationships to hyperpolarized potentials.


Fig. 2. Representative peak Ba2+ current traces recorded from Xenopus oocytes expressing the wild type and mutant Ca2+ channels. Current traces were evoked by 1400 ms depolarizing pulses from a HP of -30 to +20 mV (or +10 mV as indicated). Original current traces in the absence and he presence of 1 and 10 µM (+)S202-791 (upper panel) as well as 1 and 10 µM (-)R202-791 (lower panel) are superimposed.
[View Larger Version of this Image (14K GIF file)]


Table I.

Relative Ba2+ current amplitude through expressed Ca2+ channels in the presence of DHP agonist and antagonist

Currents were normalized to each control value. Values are the means ± S.E. of 4-20 experiments.
Wild type T1006Y T1007M Q1010M

(+)S202-791
  1 µM 3.79  ± 0.37 1.02  ± 0.05a 2.98  ± 0.24 1.14  ± 0.04a
  10 µM 4.32  ± 0.53 0.77  ± 0.05a 3.66  ± 0.32 1.65  ± 0.09a
(-)R202-791
  1 µM 0.39  ± 0.05 1.07  ± 0.06a 0.60  ± 0.05a 0.89  ± 0.04a
  10 µM 0.17  ± 0.04 0.76  ± 0.06a 0.23  ± 0.03 0.63  ± 0.08a

a  p < 0.05 versus wild type.


Fig. 3. Representative current-voltage relationships of wild type and mutant Ca2+ channels. Currents were elicited by 1400 ms voltage steps from a HP of -30 mV and recorded in the absence (open circle ) and the presence (bullet ) of 1 µM (+)S202-791 (A) as well as 1 µM (-)R202-791 (B).
[View Larger Version of this Image (14K GIF file)]


DHP antagonist activity increases when more channels are shifted to inactivated states (21). It is possible that the mutations change the distribution of Ca2+ channels active in a certain conformation. The decrease in DHP binding affinity that was determined at 0 mV membrane potential may be due to a loss of a channel's ability to become inactivated. However, our data showed that these mutants had normal basic biophysical properties. In addition, these mutants responded to DHP agonists and antagonists in parallel, even though the DHP agonist effect is not dependent on membrane potential. We believe that the decrease in binding affinity of the DHPs to these mutants is caused by a direct alteration at the interaction site between the DHP and the Ca2+ channel and not a general nonspecific conformational change. If the latter were true, we would expect electrophysiological changes in the basic properties of the channel.

In our study, we used a mammalian cell line for the binding assay and Xenopus oocytes for electrophysiological measurements. One must therefore apply caution in comparing binding dissociation constants and EC50/IC50 values. In the study of quinidine's effect on cloned K+ channels, it was reported that at least a ten times higher drug concentration was needed to achieve the same effect in the oocyte system than in mammalian cells; however, normal sensitivity was seen in excised patches (22). In mammalian cells, the IC50 of alpha 1C to DHP antagonists is approximately 10 nM. Considering this factor, our results from the oocyte system are in good agreement with the data obtained from mammalian cells in which the binding was done. In addition, our electrophysiological measurements were performed at -30 mV holding potential in which some channels (~30%) will be in the inactivated state. Thus, the results under these conditions should be reasonably comparable with those obtained from the binding studies that were performed at 0 membrane potential.

PN200-110 is an intrinsically photoactive DHP compound. The benzofurazan group at the para-position of the DHP ring can cross-link with amino acids. Photoaffinity labeling studies using PN200-110 showed that this DHP was mainly incorporated into motif III S6 (4). This suggests that the benzofurazan group of PN200-100 may associate with III S6. It is still not clear where the dihydropyridine ring binds. Radioligand binding studies suggest a common binding site for both DHP agonists and antagonists (8), whereas electrophysiological studies support the concept of different sites or different conformations of the same site (7, 9, 10). It is possible that the DHP ring of both agonists and antagonists bind to the same site but the 4-aryl ring interacts with a different component of the channel. The Q1010M and T1006Y mutant channels had a decreased sensitivity to agonists and antagonists and to a similar degree for both. Therefore, we now need to consider the importance of III S5 as being involved in the interaction with a domain shared between DHP agonists and antagonists.

Dihydropyridines bind to and modulate L-VDCC functions in a highly specific, voltage-dependent manner. Mutagenesis studies revealed a complex binding site that also emphasizes the importance of cooperative interactions. It is intriguing to compare these data with the ones obtained studying the binding site of local anesthetics on the Na+ channel (23, 24). The hydrophobic portion of local anesthetics binds to IV S6, whereas the charged portion may interact with the P regions of motifs II and III (25). Additionally, mutations that are characteristic of the disease paramyotonia congenita also modulate local anesthetic effects (LIII-IV, IV S2, and IV S4) (26). The binding site of DHPs on the L-type Ca2+ channels may be composed of III S5, III S6, and IV S6. Additional regions that appear to influence some aspects of DHP binding are III S2 and IV S3 (voltage dependence) (27) and the motif III P-region (Ca2+ dependence) (28, 29). The complexity of drug binding sites and regions that modulate drug effects indicate that these drugs bind to a "dynamic pocket" in the channel, and one must be alert to monitor several aspects of channel function to be certain that the mutations under investigation actually alter one aspect and not all of them.


FOOTNOTES

*   This work was supported in part by National Institutes of Health Grants P01-HL22619, T32 HL07382, and 5R37 HL43231 (to A. S.) and the Tanabe Seiyaku Fund for Molecular Biophysics and Pharmacology (to A. S.) The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Dagger    To whom correspondence should be addressed: Inst. of Molecular Pharmacology and Biophysics, University of Cincinnati, 231 Bethesda Ave., P. O. Box 670828, Cincinnati, OH 45267-0828. Tel.: 513-558-2200; Fax: 513-558-1778; E-mail: schwara{at}email.uc.edu.
1    The abbreviations used are: VDCC, voltage-dependent Ca2+ channels; L-VDCC, L-type VDCC; S202-791: S(+)-4(2,1,3-benzoxadiazol-4-yl)-1,4-dihydro-2,6-dimethyl-5-nitro-3-pyridincarboxylic acid isopropylester; PN200-110: isopropyl-4-(2,1,3-benzoxadiazol-4-yl)-1,4-dihydro-(2,6-dimethyl-5-methoxycarbonyl)pyridine-3-carboxylate; R202-791, R(-)-4(2,1,3-benzoxadiazol-4-yl)-1,4-dihydro-2,6-dimethyl-5-nitro-3-pyridincarboxylic acid isopropylester; DHP, dihydropyridine; PCR, polymerase chain reaction; HP, holding potential.

Acknowledgments

We thank Hiroshi Yamaguchi, Howard Motoike, and Sheryl Koch for suggestions and help during the construction of the manuscript.


Note Added in Proof

After submitting this manuscript (November 27, 1996), a paper appeared in the Journal of Biological Chemistry by Mitterdorfer et al. (Mitterdorfer, J., Wang, Z., Sinnegger, M. J., Hering, S., Striessnig, J., Grabner, M., and Glossmann, H. (1996) J. Biol. Chem. 271, 30330-30335), using molecular reagents we supplied in part. Point mutations in motif III S5 of a brain channel (BI, alpha 1A), engineered to be DHP-sensitive, using an oocyte expression system, revealed the importance of the same threonine and glutamine residues described in our paper.


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