From the
Voltage-dependent inhibition by 1,4-dihydropyridines is a
characteristic property of L-type Ca
1,4-Dihydropyridines (DHPs)
Mutant
Ca
Compared to the
wild-type
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
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 = S
The nucleotide
sequence(s) reported in this paper has been submitted to the
GenBank
We thank F. Hofmann and V. Flockerzi (Munich) for a
gift of clones of
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
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.
(
)
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.
The full-length Subunit cDNA Splice Variants
Preparation
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.
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 Ca
Xenopus laevis oocytes were
injected with 50 nl of a mixture containing cRNAs (0.5 µg/µl)
for an Channels in
Xenopus Oocytes
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).
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 V
was 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.
, 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.
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
log( 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.
/EMBL Data Bank with accession number(s) Z34810
(pHLCC70), Z34814 (pHLCC76), and Z34815 (pHLCC77).
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.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.