©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Different Voltage-dependent Inhibition by Dihydropyridines of Human Ca Channel Splice Variants (*)

Nikolai M. Soldatov , Alexandre Bouron , Harald Reuter (§)

From the (1) Department of Pharmacology, University of Bern, CH-3010 Bern, Switzerland

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Voltage-dependent inhibition by 1,4-dihydropyridines is a characteristic property of L-type Ca channels. Six out of 50 exons of the channel subunit gene are subjected to alternative splicing, thus generating channel isoform diversity. Using Xenopus oocytes as an expression system, we have found that transmembrane segment IIIS2 of human subunit is involved in the control of voltage dependence of dihydropyridine action. This segment is genetically regulated through alternative splicing of exons 21/22. Site-directed mutagenesis points to two amino acids in IIIS2, which determine the difference of the splice variants in their sensitivities to dihydropyridines. This finding provides new insight into molecular mechanisms of Ca channel inhibition by this important class of drugs.


INTRODUCTION

1,4-Dihydropyridines (DHPs)() are clinically important drugs acting on L-type Ca channels. The inhibitory potency of DHPs on these channels depends on membrane potential (1, 2) , thus indicating the complex nature of drug-receptor interaction. The structural basis of the channel for the voltage-dependent effect of DHPs is largely unknown. L-type Ca channels are oligomeric complexes composed of , , , and disulfide-linked / subunits (3) . There are three different DHP-binding subunits, , , and , generated by separate genes (4) . The channel-forming subunit is encoded by at least 50 exons (5) , and its expression is regulated through alternative splicing (6, 7) . Two exons (21 and 22), encoding the outer portion of the putative transmembrane segment IIIS2, are spliced in alternative manner. Using Xenopus oocyte as an expression system, we have found that natural splice variants of the human fibroblast subunit, containing either exon 21 or 22, differ in the voltage dependence of DHP action. Site-directed mutagenesis shows that there are two amino acids in segment IIIS2 that account for the effect.


MATERIALS AND METHODS

Subunit cDNA Splice Variants Preparation

The full-length subunit cDNA splice variants composed of exons 1-20, 21 or 22, 23-30, 33-44, and 46-50 were obtained using the HindIII/ BamHI sites of the pBluescript SK(-) vector (Stratagene) flanked at the 5`-end with HindIII/ BglII and at the 3`-end with BglII/ BamHI fragments of the Xenopus -globin gene UTR sequences, respectively (8, 9) . The recombinant plasmid pHLCC70, encoding , was constructed by sequential ligation of the following fragments of human fibroblast Ca channel cDNAs (partial clones and nucleotide positions refer to human fibroblast Ca channel cDNA (10) ): blunt BglII (vector), blunt NcoI (313) / MunI (721) from 5` (2) 6, MunI (721) / Sse8387I (2055) from f135, Sse8387I (2055)/ StyI (2721) from f28, StyI (2721)/ StyI (3420) from uf14-2, StyI (3420)/ MroI (3462) from f28, MroI (3462)/ SfuI (3726) from f39, SfuI (3726)/ SphI (4747) from h2.05, SphI (4747) NcoI (6045) from f53, NcoI (6045)/ AvrII (6419) from uf51, AvrII (6419)/ HpaI (7082) from 3t-12 and blunt BglII (vector). To construct pHLCC77 encoding , exon 21 was removed from f28, which contains both exons 21 and 22 (10) , by digesting with EcoNI (3106)/ AvrII (3181) and subsequently ligating with the sense (5`-TTCAGGAACCATATC-3`) and antisense (5`-CTAGGATATGGTTCCTGA-3`) oligonucleotides. The Sse8387I (2055)/ MroI (3462) fragment of the resulting cDNA was then used as described above. The recombinant plasmid pHLCC76 encoding was constructed using the NsiI (4315)/ CelII (4487) fragment of uf20-1 cDNA in sequential ligations leading to pHLCC77.

Mutant Ca channel plasmid pHLCC77c (Y958I) was prepared by the two mutagenic primers method (11) using the respective wild type channel-encoding plasmids and 32-mer oligonucleotides carrying the desired double base mismatches (5`-CTGGAACGAGTGGAAATTCTCTTTCTCATAAT-3` and 5`-TAGGCAATGCAGACATTGTCTTCACTAGTATC-3`). Mutants pHLCC77p (G954F) and pHLCC77cp (G954F,Y958I) were made using the polymerase chain reaction technique (Hoffman-LaRoche) within the EcoNI (3106)/ SfuI (3726) cassette with the sense primer 5`-ACCTCCTTCAGGAACCATATCCTATTCAATGCAG-3` and pHLCC77 or pHLCC77c as templates. Nucleotide sequences of all obtained cDNAs were verified by the modified dideoxy termination method (12) . Template DNAs were linearized by digestion with BamHI, and capped transcripts were synthesized in vitro with T7 RNA polymerase using the mRNA cap kit (Stratagene). Before injection, mRNA samples were dissolved in 5 mM HEPES, pH 6.8.

Expression of CaChannels in Xenopus Oocytes

Xenopus laevis oocytes were injected with 50 nl of a mixture containing cRNAs (0.5 µg/µl) for an isoform, (13) , and (14, 15) subunits at 1:1:1 molar ratio. Oocytes were defolliculated 1-3 days before the measurements. The two-electrode voltage clamp method (Axoclamp 2-A amplifier) were used to record whole cell Ba currents. Electrodes filled with 3 M KCl, 0.1 mM EGTA solution had resistances between 0.2 and 1 megaohms. The external solution contained 40 mM Ba(OH), 50 mM NaOH, 1 mM KOH, and 10 mM HEPES (pH 7.45 with methanesulfonic acid). Voltage clamp protocols and current measurements were commanded and analyzed by using the Cambridge Electronics Design (EPC) software running on a microcomputer. Membrane currents, filtered at 0.5-1 KHz and sampled at 2-4 KHz, were triggered by 1-s step depolarizations applied from holding potential of -90 or -40 mV. After an equilibration period of 20-30 min, stable Ba currents could be recorded for up to 80 min. A slow stimulation frequency (0.05 Hz) was essential to achieve complete recovery from inactivation occurring during the pulse. Drugs were added to the superfusion medium, and currents were monitored until a steady state was reached. All experiments were performed at room temperature (20-22 °C).


RESULTS AND DISCUSSION

Exons 21/22 Are Involved in Voltage Dependence of DHP Action

We have prepared three natural splice variants of the human L-type Ca channel subunit (10) . Either exon 21 () or 22 () were selectively incorporated into the nucleotide sequence (Fig. 1 A) between invariant exons 20 and 23. Exon 33 was deleted in . The respective cRNAs, when co-injected with regulatory subunits (13) and (14, 15) into Xenopus oocytes, gave rise to expression of Ca channels. Time courses, ranges of activation and inactivation, as well as the peaks of Ba currents through these channels were very similar in all splice variants (Figs. 1 C and 2).


Figure 1: Transmembrane segment IIIS2 encoded by alternative exons 21 or 22 represents a modulatory site for 1,4-dihydropyridine blockers of L-type Ca channels. A, a putative transmembrane folding of the subunit of L-type Ca channel is shown at the top. The model depicts four repetitive motifs of homology ( I-IV), each composed of six transmembrane segments (S1 S6). It also shows portions of segment IIIS2 ( dark cylinder) and IVS3 ( striped cylinder) modulated through alternative splicing. Three cDNA constructs encoding splice variants are shown below as insertions ( open rectangles) into modified pBluescript SK(-) vector, flanked by 5`- and 3`-UTRs from the Xenopus globin gene ( solid rectangles). Vertical bars with numbers (21, 22, 31-33) indicate positions and sequential numbers of alternative exons. Deleted exons are indicated by interruptions in open rectangles and are listed on the right together with names of splice variants referred to throughout the text. Names of expression plasmids as reported to the EMBL DNA data bank are shown on the left of each construct. B, alignment of amino acid sequences encoded by exons 21 and 22. Amino acid differences are shaded. Nonequivalent substitutions studied by site-directed mutagenesis are boxed; the horizontal bar indicates the putative transmembrane segment IIIS2. C, representative Ba currents evoked by step depolarizations to 0, +20, and +40 mV from a holding potential of -90 mV ( upper panel) and current-voltage relationships of peak Ba current ( lower panel).



Voltage dependence of the inhibitory effect of the DHP derivative (+)-isradipine on Ba currents (2) was studied by activating the current with step depolarizations from two holding potentials ( V= -40 mV and -90 mV). When elicited from V= -40 mV, Ba current through and channels was inhibited by (+)-isradipine in the same concentration range (Fig. 2, A and B, ). However, at the holding potential of -90 mV, the slope of the concentration-response curve was significantly less steep in than in , causing an 8.6-fold difference in the inhibitory potency of the drug between the two channels (Fig. 2, A and B, ). These results suggest that the external portion of the putative transmembrane segments IIIS2, encoded by exons 21 and 22, experiences voltage-dependent conformational changes, which affect DHP binding. The voltage dependence of action of isradipine is much more pronounced in channel than in . This difference is not due to different extents of steady state inactivation of and , which was found to be the same when Vwas changed from -90 to -40 mV (39.8 ± 2% and 37.9 ± 2%). So far, we have no detailed information whether on- or off-rates of drug effects are different at V= -90 mV.


Figure 2: Traces of Ba current through ( A), ( B), and ( C), and concentration-response curves for the block of Ca channels by (+)-isradipine. Oocytes were voltage-clamped from a holding potential of -40 mV ( left panel) or -90 mV ( middle panel) to +20 mV. Ba current traces illustrate the responses obtained in the absence ( Cont) and presence of (+)-isradipine (50 or 1000 nM). Averaged concentration-response curves ( right panels) of fractional inhibition of Ba current were measured in the presence of 5, 10, and 50 nM (+)-isradipine at V = -40 mV () and 50, 200, and 1000 nM (+)-isradipine at V = -90 mV (). Values are means ± S.E. of 4-12 oocytes.



Our data show that the amino acid sequence, encoded by exons 21/22, represents a new, previously unknown modulatory site, which is different from all earlier proposed DHP binding sites (16, 17, 18) . Close to the IVS5-IVS6 region shown to be critical for the DHP sensitivity of (18) is the external linker between segments IVS3 and IVS4. In a number of transcripts, this linker is shortened due to deletion of combinatorial alternative exon 33 (6, 10, 19, 20) . In order to find out whether this linker is important for the DHP action, we have prepared the channel splice variant , where exon 33 was deleted from the coding frame. DHP inhibition and kinetic parameters of Ba currents of the respective channel splice variant were similar to those of (Fig. 2 C and ). Therefore, the IVS3-IVS4 linker contributes neither to DHP action nor to the channel kinetics.

Structural Basis for the DHP Binding Modulatory Site

Splice variants and differ in only seven uncharged amino acid residues (Fig. 1 B). Five substitutions are equivalent and do not change the hydropathicity of the IIIS2 segment. However, there are two hydrophobic residues, Phe and Ile in , instead of hydrophilic residues Gly and Tyr in the splice variant . This difference may affect voltage-dependent inhibition of the Ca channel by (+)-isradipine. Therefore, we have prepared mutants of with incorporated substitution of G954F, Y958I, or both.

Compared to the wild-type , single mutations, G954F or Y958I, reduced the slopes of the concentration-response curves at holding potential of -90 mV only slightly ( and Fig. 3 ). However, double mutation (G954F,Y958I) in caused voltage-dependent inhibition by (+)-isradipine which resembled that of . At V= -90 mV, the slope of the concentration-response curve decreased and became similar to that of . Compared to , the curve was slightly shifted to the left, and the IC increased 3.8-fold ( Fig. 3and ). By contrast, at V= -40 mV, slopes and IC were the same in all constructs ( Fig. 3 and ). Whether the difference in IC values between the double mutant and at V = -90 mV is due to other amino acids in exons 21/22 remains to be seen.


Figure 3: Concentration-response curves of inhibition of wild-type and mutant Ca channels by (+)-isradipine. Oocytes were voltage-clamped from a holding potential of -90 mV ( A) and -40 mV ( B) to +20 mV. , wild-type ; , ; , ; , ; , wild-type . Values are means ± S.E.; numbers of oocytes are given in Table I. Compared to (), the mutated Ca channels () and () show similar DHP sensitivity ( A and B). However, at -90 mV ( A), the double mutant () is significantly less DHP-sensitive than () and behaves like (). The fraction of Ba current not inhibited by 1000 nM (+)-isradipine at V = -90 mV was 0.23 ± 0.03 for () and 0.43 ± 0.03 ( p < 0.01) for ().



Both alternative amino acid sequences of segment IIIS2 contain three invariant charged residues, Asp, Glu, and Lys, which are also conserved in other Ca channels. These residues may form salt bridges with charged amino acids at adjacent transmembrane segments (21, 22) and are potentially susceptible to transmembrane voltage. The position of these three residues may change within the protein when the hydrophobicity of the IIIS2 segment is altered by substitution in exons 21/22. Such changes could affect cooperative arrangements of their electrostatic interactions and, thus, the transition from low to high affinity conformation of the DHP binding site when holding potential was changed from -90 to -40 mV. Substitution of hydrophilic amino acids in positions 954 and 958 of for hydrophobic ones is an important factor in the reduction of voltage-dependent inhibition of by DHPs.

Since the difference in voltage sensitivity of DHP action shown in this study is a property of naturally occurring splice variants of human L-type Ca channels, it may contribute to the tissue specificity of this class of drugs (23, 24) . Our results also show the complexity of drug-receptor interactions. It is clear that the amino acid sequence in IIIS2 is not the primary binding site for DHPs. Since the affinities of all constructs for the drug are identical at a holding potential of -40 mV, but very different at V= -90 mV, multiple modulatory sites seem to affect binding affinities of DHPs in a voltage-dependent manner.

  
Table: Concentration dependence of Ba current inhibition by (+)-isradipine measured at holding potentials of -90 and -40 mV

Slopes of the regression line, S, (means ± S.E.) were calculated by logarithmic fitting of the concentration-response curves obtained in individual oocytes as y = Slog( x) + c, where y is the fraction of noninhibited current, x is the concentration of (+)-isradipine, and c is a constant. IC values were estimated graphically from the regression lines; n, numbers of oocytes tested.



FOOTNOTES

*
This work was supported by Swiss National Science Foundation Grant 31-29862.90 and a grant from the Sandoz Foundation. 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.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank/EMBL Data Bank with accession number(s) Z34810 (pHLCC70), Z34814 (pHLCC76), and Z34815 (pHLCC77).

§
To whom correspondence and reprint requests should be addressed: Dept. of Pharmacology, University of Bern, Friedbühlstrasse 49, CH-3010 Bern, Switzerland. Tel.: 41-31-632-3281; Fax: 41-31-302-7230.

The abbreviations used are: DHP, dihydropyridine; V, holding potential.


ACKNOWLEDGEMENTS

We thank F. Hofmann and V. Flockerzi (Munich) for a gift of clones of and subunits, J. Tytgat (Leuven) for providing pGEMHE, E. Sigel, R. Bauer, and A. Cachelin for helpful advice, H. Porzig and R. Zühlke for reading the manuscript, and Heleen van Hees for excellent technical assistance.


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©1995 by The American Society for Biochemistry and Molecular Biology, Inc.