(Received for publication, December 2, 1996)
From the Institute of Molecular Pharmacology and Biophysics, University of Cincinnati, Cincinnati, Ohio 45267-0828
The 1 subunit of L-type
voltage-dependent Ca2+ channels
(
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
1C, changing the amino acids to their
counterparts in the DHP-insensitive
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.
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 1 and the
DHP-insensitive VDCC
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
1C channel into the
1A channel (10). This
alteration was sufficient to confer sensitivity to both DHP agonist and
antagonist to the
1A channel.
At the same time, we found that by replacing the III S5-S6 of the
1C with that of the corresponding region of
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
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
1C channel (12, 13) to those in the
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
1 subunit of the
L-VDCC.
The wild
type and mutant rabbit heart L-VDCC 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
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
1 cDNAs were transferred from pBluescript II SK(
)
into the mammalian expression vector pAGS-3 (15) at HindIII
and NotI sites.
HEK 293 cells were transfected with 1,
2/
a (16), and human
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).
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 ExpressionExpression of the wild type and
mutant calcium channels in oocytes was done as described previously
(7). In order to achieve maximal expression, the 1c
subunit cRNAs were coinjected with cRNAs specific for the
2/
a (16) and human
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:
![]() |
(Eq. 1) |
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 AnalysisThe 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.
Our previous work investigated the contribution of motif IV to DHP
effects by testing chimeric 1 subunits engineered
between the DHP-sensitive rabbit heart L-VDCC (Ca9) and the
DHP-insensitive brain BI-2 (
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
1A, DHP sensitivity was lost.
A two-pronged experimental approach was launched. Following
site-directed mutagenesis to change nonconserved amino acids in 1C (Ca9) into the corresponding amino acids of
1A subunit, each mutant was transiently coexpressed in
HEK 293 cells with the accessory
2/
a and
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
-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
-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."
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 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
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.
|
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 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.
We thank Hiroshi Yamaguchi, Howard Motoike, and Sheryl Koch for suggestions and help during the construction of the manuscript.
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, 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.