From the Institut für Biochemische Pharmakologie, Peter-Maystrasse 1, A-6020 Innsbruck, Austria
Received for publication, November 8, 2000, and in revised form, January 12, 2001
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ABSTRACT |
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We investigated the mechanism of
interaction of individual L-type channel amino acid residues with
dihydropyridines within a dihydropyridine-sensitive The membrane depolarization-dependent opening of
voltage-gated Ca2+ channels effectively modulates
Ca2+ influx into electrically excitable cells. These
channels represent key elements in Ca2+ signaling,
controlling neurotransmitter release, synaptic plasticity, muscle
contraction, and pacemaker functions. So far selective nonpeptide
Ca2+ channel modulators have only been developed for L-type
Ca2+ channels which are expressed primarily in the
cardiovascular system but also in neuronal and neuroendocrine cells (1,
2). Other Ca2+ channel types, such as N, P, Q, and R-type
Ca2+ channels, play a prominent role for fast
neurotransmitter release in neurons (1), but they are insensitive to
L-type Ca2+ channel modulators. 1,4-Dihydropyridines
(DHPs),1 such as isradipine
or amlodipine, are very well characterized L-type Ca2+
channel blockers ("antagonists") that are also used to treat cardiovascular diseases. In contrast, DHP Ca2+ channel
activators ("agonists," e.g. BayK8644) stimulate
Ca2+ currents through L-type channels. DHPs do not modulate
ion currents through, e.g. P- or Q-type channels.
This difference in DHP sensitivity is the result of different Individual amino acid residues that form the DHP binding pocket in
L-type Although site-directed mutagenesis has been used to identify residues
important for DHP interaction, the exact role of some of these residues
in antagonist and agonist interaction has not been completely studied.
In addition, for some of these L-type residues it is still unclear if
their introduction into To address this question we used site-directed mutagenesis to study
further the mechanism of DHP interaction with L-type residues in repeat
III of a DHP-sensitive Construction of Mutant
Mutations of Thr1393 and Gln1397 (
Mutagenic primers contained silent point mutations to introduce or
eliminate restriction endonuclease sites for verification of the
desired mutations. Fragments amplified by PCR were sequenced entirely
to confirm sequence integrity.
Expression of Electrophysiological Measurements--
1-6 days after cRNA
injection inward barium currents (IBa)
through voltage-gated Ca2+ channels were measured at room
temperature using the two-microelectrode voltage-clamp technique as
described previously (9). To quantifiy endogenous
IBa X. laevis oocytes injected only
with
The extracellular solution contained 40 mM
Ba(OH)2, 50 mM NaOH, 2 mM CsOH, and
5 mM HEPES (pH adjusted to 7.4 with methanesulfonic acid).
The voltage recording and current-injecting microelectrodes were filled
with 2.8 M CsCl, 0.2 M CsOH, 10 mM
HEPES, and 10 mM EGTA (adjusted to pH 7.4 with HCl) and had
resistances of 0.7-6 megohms.
Modulation of peak IBa was measured from the
indicated standard holding potentials to a test pulse corresponding to
the peak potential (IBa block by DHP
antagonists) or 10 mV positive to the peak potential of the
current-voltage relations (IBa stimulation by
DHP agonist and FPL64176, respectively). Standard pulse frequency was
0.017 or 0.034 Hz to assess channel modulation. Isradipine was employed
at a concentration (10 µM) causing near complete channel
block in oocytes at negative holding potentials (6).
The time course of peak current inhibition by isradipine was estimated
by fitting the peak currents of successive episodes to a
monoexponential function (IBa = A*exp(
Because of slow recovery from inactivation, steady-state inactivation
was quantified from the decline of peak IBa
after switching from a holding potential of Reagents--
(±)-Isradipine (PN200-110) was from Sandoz AG
(Basel, Switzerland), (±)-BayK8644 was from Bayer AG (Wuppertal,
Germany), and FPL64176 was from Fisons Pharmaceuticals (Leicestershire,
U. K.). Drug-containing solutions were freshly prepared from 10 mM stock solutions (in dimethyl sulfoxide) and applied at
the same flow rate as control solution.
Statistics--
Data are given as the mean ± S.D.
Statistical significance was calculated using the unpaired Student's
t test, employing OriginR (Microcal, version
6.0).
We and others have shown recently that 1A subunit
(
1ADHP). Mutation of individual residues in repeat
III and expression in Xenopus oocytes revealed that
Thr1393 is not required for dihydropyridine interaction but
that bulky side chains (tyrosine, phenylalanine) in this position
sterically inhibit dihydropyridine coordination. In position 1397 a side chain carbonyl group was required for high antagonist
sensitivity. Agonist function required the complete amide group of a
glutamine residue. Val1516 and Met1512 side
chains were required for agonist (Val1516) and antagonist
(Val1516, Met1512) sensitivity. Replacement of
Ile1504 and Ile1507 by
1A phenylalanines was
tolerated. Substitution of Thr1393 by phenylalanine or
Val1516 by alanine introduced voltage dependence of
antagonist action into
1ADHP, suggesting that these
residues form part of a mechanism mediating voltage dependence of
dihydropyridine sensitivity. Our data provide important insight into
dihydropyridine binding to
1ADHP which could facilitate
the development of
1A-selective modulators. By modulating P/Q-type
Ca2+ channels such drugs could serve as new anti-migraine therapeutics.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1
subunit isoforms which, together with accessory subunits such as
2-
and
, form the Ca2+ channel complexes. L-type
Ca2+ channels are formed, e.g. by DHP-sensitive
1C or
1S subunits, whereas P- and Q-type Ca2+
channels contain DHP-insensitive
1A subunits.
1 subunits have recently been identified (for review, see
Refs. 3-5). Most interestingly, some but not all of these residues are
conserved in non-DHP-sensitive subunits, such as
1A and
1E.
These subunits are rendered fully DHP agonist- and antagonist-sensitive
after introduction of the remaining nonconserved L-type residues
forming the binding pocket (yielding, e.g. DHP-sensitive
1A subunits,
1ADHP (6, 7)).
1A creates additional interaction sites for
the DHP molecule or removes an inhibitory effect of the corresponding
1A residue. A more detailed insight into the molecular mechanisms of
DHP binding to its recombinant drug binding domain in
1ADHP could provide important structural information for
developing small molecules with affinity for
1A subunits. Such drugs
could be useful therapeutics for the treatment of
1A-associated
disorders such as migraine (8).
1A subunit (
1ADHP). By
carrying out these studies within the
1A background our results can
be applied to future studies aimed at the development of
1A-selective Ca2+ channel modulators.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1 cDNAs--
The construction
of
1ADHP and M1512V
(
1ADHPi) was described previously (6). Mutations were
introduced into
1ADHP cDNA containing silent
restriction sites (indicated by asterisks) generated by polymerase
chain reaction (PCR) in previous cloning steps (9). Mutant
1 cDNAs were created by PCR using the "gene
SOEing" technique (10) employing proofreading Pfu
polymerase (Stratagene).
1A
numbering (11); corresponding to positions 1039 and 1043 in
1C-II,
respectively (12)) were introduced by PCR using appropriately mutated
SOE-PCR primers into a SfiI-SalI* cassette
(nucleotides 4297-4744,
1A numbering) of an
XhoI-ClaI* fragment (nucleotides 1689-4925,
domains II and III) of
1ADHP subcloned into pBluescript
SK+ (Stratagene). The mutations were reintroduced through
an NheI-ClaI* fragment (nucleotides 3543-4925,
domain III) into
1ADHP in PNKS2 (provided by
O. Pongs, ZFMNB, Hamburg, Germany), thus yielding the following
1ADHP mutants: T1393A, T1393Y, T1393F, T1393S, Q1397A,
Q1397M, Q1397E, and Q1397N. Mutations in transmembrane segment IIIS6
were introduced by PCR into an SalI*-ClaI*
cassette (nucleotides 4744-4925) of
1ADHP in
PNKS2, thus yielding the following
1ADHP
mutants: Y1503A, I1504A, I1504F, I1507A, I1507F, P1508A, M1512A, and
V1516A, corresponding to positions 1152, 1153, 1156, 1157, 1161, and
1165 in
1C-II, respectively.
1 Mutants in Xenopus laevis Oocytes--
Capped
run-off poly(A+) cRNA transcripts from
XbaI-linearized cDNA templates were synthesized
according to Krieg and Melton (13).
1 cRNA was coinjected with
1a
(14) and
2
(15) subunit cRNAs into stage V-VI oocytes from
X. laevis.
1a and
2
were analyzed in parallel. Only oocytes
expressing peak IBa through recombinant
Ca2+ channels at least three times as high (usually >100
nA) as the highest endogenous currents were included into analysis.
Data analysis and acquisition were performed by using the pClamp
software package (version 6.0, Axon Instruments). Leakage correction
was performed by adjusting the current traces by a factor calculated from the difference between the leak at
80 mV and
90 mV, respectively.
t/
) + C).
120 mV to more positive
holding potentials allowing equilibrium IBa to
be reached. For mutant V1516A, which showed relatively little
inactivation at
80 mV,
100 mV was used as reference potential for
the resting state.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1A
(
1ADHP, Fig.
1A) and
1E subunits become
highly DHP-sensitive by mutation of 8-9 amino acids to residues
present in L-type
1 subunits (6, 7, 16). In this study we used
1ADHP as a suitable model to study further the mechanism
by which amino acid residues in transmembrane segments IIIS5 and IIIS6
participate in DHP interaction. The
1A structural background should
allow application of the results for
1A drug development.
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Fig. 1.
Effects of single mutations in IIIS5 on DHP
sensitivity. A, schematic representation of
1ADHP. L-type residues (black
circles) are given in single-letter code. B, amino acid
sequence alignment of
1C,
1A,
1ADHP, and
1ADHP mutants. Nonconserved L-type channel amino acids
(black boxes) and substituted amino acids in mutants of
1ADHP (gray boxes) are highlighted.
C, isradipine (10 µM) inhibition and BayK8644
(10 µM) stimulation of peak IBa
through mutant Ca2+ channels expressed in X. laevis oocytes. Drug effects were determined at different holding
potentials (black bars,
80 mV; white
bars,
100 mV). Data are presented as the means ± S.D. from the indicated number of experiments; statistically
significant differences from
1ADHP (
80 mV) are
indicated: *, p < 0.05; **, p < 0.01.
Role of Thr1393 in Transmembrane Helix
IIIS5--
Previous studies have shown that replacement of the IIIS5
residues Thr1393 or Gln1397 (numbering
according to 1ADHP, see also Fig. 4) in DHP-sensitive
1A chimeras (17) or
1C (18) by the corresponding
1A residues (T1393Y, Q1397M) substantially decreased DHP sensitivity. It is unclear
if this is because of a steric interference introduced by the
corresponding tyrosine or methionine side chain, respectively, or the
result of the removal of the appropriate pharmacophores. To address
this question we replaced Thr1393 or Gln1397 in
1ADHP by a series of other residues and tested the
consequences of these mutations for modulation of
IBa by the DHP Ca2+ channel blocker
isradipine and activator BayK8644 after expression in
Xenopus oocytes. In agreement with previous results, 10 µM isradipine inhibited 72.1 ± 6.9%
(n = 4, Fig. 1C) of
IBa through
1ADHP elicited from a
holding potential of
80 mV. 10 µM BayK8644 stimulated
IBa by 4.3 ± 1.2-fold (n = 4). Sensitivity was abolished after mutation of Thr1393 to
tyrosine (mutant T1393Y: 4 ± 8.3% inhibition, n = 5; 1.4 ± 0.7-fold stimulation, n = 7). T1393Y
was also insensitive to stimulation by the non-DHP agonist FPL64176
(1.04 ± 0.1 fold stimulation, n = 3). Fig.
1C shows that reduction of the side chain size by mutation
from threonine to serine (mutant T1393S: 59.2 ± 16.3% inhibition, n = 3; 3.6 ± 0.4-fold stimulation,
n = 5; p > 0.05) or alanine (mutant
T1393A: 59.5 ± 10.3% inhibition, n = 5; 5.6 ± 2.8-fold stimulation, n = 4, p > 0.05) not only preserved full DHP antagonist sensitivity but also
supported stimulation by BayK8644. These results show that the side
chain of Thr1393 is not essential for DHP modulation.
Instead, the bulky hydroxyphenyl moiety of tyrosine must prevent DHP
interaction (19).
To determine whether DHP antagonist sensitivity was affected by
membrane voltage, inhibition by isradipine was measured also at 100
mV holding potential at which a much smaller fraction of channels
underwent steady-state inactivation than at
80 mV (Table
I). As for
1ADHP (Fig. 2;
20), DHP sensitivity was not affected by membrane voltage in T1393A
(
100 mV: 62.1 ± 10, 5% inhibition, n = 4) and
T1393Y (Fig. 2;). No significant
antagonist sensitivity of T1393Y was recovered at holding potentials
causing 70% steady-state inactivation (12.0 ± 13.2%,
n = 3; Fig. 2). Steady-state inactivation properties of
mutant T1393Y were similar to
1ADHP (Table I). This
rules out decreased antagonist sensitivity of T1393Y being caused by
changes in channel inactivation properties.
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Next we investigated whether the hydroxyl group of the tyrosine side
chain contributes to the reduction of DHP sensitivity in T1393Y by
creating mutant T1393F. As illustrated in Fig. 2, the isradipine
sensitivity of peak IBa through T1393F was
reduced dramatically at 100 mV holding potential (8.8 ± 5.0%
inhibition, n = 3), and stimulation by BayK8644 was
negligible (1.34 ± 0.28-fold stimulation, n = 3, Fig. 1). However, isradipine increased IBa decay
during depolarization causing 42 ± 20% (n = 3)
inhibition at the end of the 350-ms test pulse (Fig. 2A).
This indicated that the drug still interacts with depolarized channels.
The increase in IBa decay may be caused by
preferential interaction with or promotion of inactivated channel
states (20) and prompted us to study DHP block at more depolarized
holding potentials. Unlike T1393Y, DHP antagonist sensitivity of T1393F
increased substantially at more positive holding potentials, and
inhibition of peak IBa was recovered completely
at potentials causing 70% steady-state inactivation (Fig. 2).
Interestingly, development of block was slowed compared with
1ADHP (T1393F:
= 4.51 ± 1.38 min,
n = 6;
1ADHP:
= 2.28 ± 0.26 min, n = 4, p < 0.05).
No evidence for a similar voltage-dependent action was
found for BayK8644 (Fig. 1C). Taken together our experiments
show that the tyrosine's phenyl ring alone is sufficient to decrease
1ADHP antagonist sensitivity at negative potentials but
that the additional hydroxyl group is required to maintain this effect
at more positive voltages.
Role of Gln1397 in IIIS5--
Previous experiments
have shown that mutation of IIIS5 Gln1397 to the
corresponding methionine (17, 18) reduces DHP sensitivity. This was
also observed after introduction of this mutation (Q1393M) into
1ADHP (Fig. 1C). Antagonist sensitivity was
decreased, and simulation by the agonists FPL64176 (0.95 ± 0.13-fold stimulation, n = 3) and BayK8644 (Fig.
1C) was abolished. In contrast to Thr1393,
introduction of an alanine, resulting in mutant Q1397A, did not support
full DHP antagonist (29.6 ± 13.5% inhibition, n = 7) and agonist (1.4 ± 0.15-fold stimulation, n = 5) sensitivity (Fig. 1C). This indicates that the side
chain of Gln1397 participates directly in DHP binding.
Replacement of glutamine by asparagine, which shortens the side chain
by one methylene group, conferred full antagonist sensitivity
(68.8 ± 9.8%, n = 5, p > 0.05)
but significantly reduced stimulation by the agonist (1.8 ± 0.66-fold, n = 5). A similar finding was obtained for
mutant Q1397E (63.5 ± 15% inhibition, n = 5;
1.8 ± 0.36-fold stimulation, n = 4) and Q1397D
(17) in which the side chain NH2 groups are replaced by OH
groups. Taken together these results confirm our previous hypothesis
that the carbonyl group (present in the amide and carboxyl side chains)
in position 1397 is sufficient for DHP antagonist sensitivity, whereas
full agonist action requires the complete amide moiety. Our data show
that the latter must even be located in an appropriate distance from
the IIIS5 backbone because only glutamine but not asparagine supports
full activity.
Role of IIIS6 Residues--
Previous radioligand binding (21) or
functional (22) studies have shown that Tyr1503,
Ile1504, Ile1507, and Met1512 are
important determinants of isradipine affinity in transmembrane helix
IIIS6. However, the contribution of Tyr1503,
Ile1504, and Ile1507 have not yet been
determined within the 1ADHP background. As shown in Fig.
3B, mutation of these residues
to alanine did not decrease antagonist sensitivity (
80 mV). This is
consistent with previous results obtained with mutated
1C subunits,
in which only 2.5-5-fold lower affinities at
80 mV were reported for
these single mutations in functional experiments (22). As in
1C
(22), mutation Y1503A, but not I1504A and I1507A, abolished agonist stimulation of peak current (Fig. 3B). The activating effect
of BayK8644 was converted into an inhibitory one, evident as inhibition of 42 ± 8.4% (n = 3) of
IBa at the end of the 350-ms test pulse (Fig.
3C). This demonstrates that the mutation does not block DHP
interaction with the channel but prevents the agonist from stabilizing
open channel conformations. Because BayK8644 is employed as the racemic
mixture of an agonist ((
)-enantiomer) and a weak antagonist
((+)-enantiomer), IBa inhibition most likely
reflects channel block by the antagonistic enantiomer in the absence of stimulation by the agonist. This is supported further by the finding of
the complete lack of IBa modulation by the
optically pure agonist FPL64176 (0.97 ± 0.12-fold stimulation,
n = 3).
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To address the question of whether DHP sensitivity is still maintained
after back-mutation of L-type residues in positions 1504 and 1507 to
the corresponding 1A phenylalanines, we constructed mutants I1504F
and I1507F. Both mutants showed only minor and nonsignificant decreases
in isradipine inhibition compared with
1ADHP. This
demonstrates that the DHP sensitivity of
1A can be achieved by
introduction of an even lower number of L-type residues into
1ADHP than described previously. Both mutants were efficiently stimulated by 10 µM BayK8644. In the case of
I1507F, stimulation was even more pronounced than for
1ADHP (15.6 ± 7.6-fold, n = 7).
Such an enhancement was also found for stimulation by FPL64176,
although not on the level of statistical significance (I1507F: 4.1 ± 1.16-fold, n = 4, Fig. 3C;
1A-DHP: 2.8 ± 0.84-fold, n = 3 (6)).
We also analyzed the requirement of the Met1512 side chain for agonist action. In agreement with earlier studies (21) mutation M1512A reduced isradipine sensitivity (Fig. 3B), suggesting that the Met1512 side chain interacts directly with the DHP antagonist. This side chain preferentially mediated antagonist effects because stimulation by BayK8644 was not affected by the mutation (5.5 ± 2.6-fold stimulation, n = 6). This is in contrast to our previous observation in which substitution of this residue by a valine reduced both agonist and antagonist sensitivity (Fig. 3B (6)).
Other residues in IIIS6 which may affect drug modulation by DHPs are
Pro1508 and Val1516. In position 1508 both
alanine and proline support high affinity DHP antagonist interaction in
1C (23) and
1ADHP (Fig. 3B). However,
mutation of Pro1508 to alanine may cause mutation-induced
conformational changes of the IIIS6 helix (24) which could affect
modulation by agonists. This possibility has not yet been investigated.
We found no evidence for such an effect in P1508A (Fig. 3B).
Val1516, which is conserved in L- and non-L-type
Ca2+ channel
1 subunits, was recently found to
contribute to Ca2+ channel block by phenylalkylamines (25)
and (+)-cis-diltiazem (26), but its contribution to DHP sensitivity is
unknown. Figs. 2 and 3 illustrate that mutation V1516A decreased
stimulation by BayK8644 and inhibition by isradipine at a
80 mV
holding potential. As for T1393F, V1516A increased
IBa decay during the test pulse. At
80 mV peak
IBa was reduced by 21 ± 12%
(n = 5), but IBa at the end of
the 350-ms test pulse was reduced by 55 ± 15% (n = 5). This mutation also introduced voltage dependence for isradipine block, as decreased DHP sensitivity was not observed at holding potentials where 70% of the channels were inactivated (Fig. 2). In
contrast to T1393F, no slowing of isradipine block development was
evident (
= 1.53 ± 0.96 min, n = 3).
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DISCUSSION |
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We have studied the contribution of residues previously found to
mediate sensitivity of L-type Ca2+ channel 1 subunits to
DHP Ca2+ channel blockers and activators. Our data
(summarized in Fig. 4) provide further structural insight into the
differential affinity of
1A and L-type
1 subunits for DHP
Ca2+ channel modulators.
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Decreased DHP sensitivity after the introduction of bulky tyrosine or
phenylalanine residues into position 1393 of 1A (T1393Y, T1393F) is
not caused by the removal of a critical binding contact. Instead, the
bulky side chains sterically prevent high affinity DHP interaction. In
T1393F, i.e. in the absence of the tyrosine hydroxyl group,
obstruction of DHP antagonist interaction is only found for resting
channels but is nearly absent when channels are inactivated. It is
tempting to speculate that the hydroxyl function stabilizes a certain
orientation of the tyrosine side chain which interferes with DHP
antagonist coordination independent of channel conformation. This may
be accomplished, e.g. by forming a hydrogen bond with other
residues. From a working model of the DHP binding domain (19) based on
the three-dimensional coordinates of the kcsA potassium channel (24)
the carbonyl oxygen of the IIIS6 Met1511 would be the most
likely candidate for such hydrogen bonding (not shown). Removal of the
hydroxyl group (in mutant T1393F) still prevents high DHP antagonist
sensitivity of resting channels but restores sensitivity when the
channels inactivate. We propose that hydrogen bonding does not occur
without the hydroxyl group, providing the phenyl group with more
rotational flexibility such that DHP antagonist binding to inactivated
states is possible. This flexibility may delay, but not prevent, DHP
association to the channel. Such a model can explain the observed
slower onset of DHP block of IBa through T1393F
compared with
1ADHP.
We present evidence that, in contrast to Thr1393, the Glu1397 side chain is involved directly in DHP binding. Maximal DHP antagonist sensitivity critically depends on the presence of a side chain carbonyl group, a potential hydrogen bond acceptor, whereas DHP agonist action requires an intact amide function spaced at a defined distance (glutamine amide) from the IIIS5 protein backbone.
Introduction of nine L-type channel amino acids into 1A
renders the resulting DHP-sensitive construct (
1ADHP)
highly sensitive to DHPs. Although
1ADHP retains most of
the hallmarks of DHP interaction with L-type Ca2+ channel
1A subunits (such as high affinity, stereoselectivity, and
allosteric modulation by non-DHP Ca2+ antagonists (19)),
the typical voltage dependence of DHP block is absent (20). This has
also been described previously for a DHP-sensitive
1E subunit
construct (27). We show that the change of only a single amino acid
residue can introduce considerable voltage dependence of the DHP block
into
1ADHP. This is an important observation because
the molecular mechanism for voltage-dependent Ca2+ channel block by DHPs is not well understood. Our
experiments clearly demonstrate that first, alterations of single amino
acid side chains affect voltage-dependent DHP block. In
previous studies the voltage dependence of
1C subunits has been
associated with sequence divergence of multiple residues within larger
sequence stretches (comprising transmembrane segment IS6 (27, 28)). Second, we found that single mutations within two distinct but adjacent
transmembrane segments of the DHP binding domain can modify the voltage
dependence of DHP inhibition of
1ADHP by apparently different mechanisms. The addition of a bulky phenylalanine
in position 1393 mainly decreases DHP antagonist sensitivity for resting channels. This steric hindrance decreases when the channel assumes the inactivated conformation (see above). A different mechanism
seems to apply for mutation V1516A. In this case, DHP sensitivity also
decreases mainly for resting channels after removal of the
valine side chain by mutation to alanine. This suggests that the valine
side chain is essential for DHP action in the resting channel
conformation but is not important once the channel inactivates. The
relative contribution of individual residues for DHP binding must
therefore change upon depolarization. We cannot distinguish whether the
Val1516 side chain interacts directly with the DHP molecule
in the resting state or contributes indirectly to a hydrophobic
stabilization of the binding domain which is disrupted by its mutation
to alanine. Taken together, our experiments identify two basic
mechanisms that explain voltage-dependent changes in DHP
sensitivity, at least in
1ADHP Ca2+
channels: gating-induced molecular rearrangements within the DHP
binding domain can alter DHP antagonist affinity either by changing the
extent of steric hindrance by a particular residue (such as in T1393F)
or by providing (or stabilizing) suitable attachment sites for the drug
(such as in V1516A).
Our results contribute important information for the development
of P/Q-type Ca2+ channel-selective modulators. DHP
derivatives would represent ideal lead compounds for the development of
1A Ca2+ channel modulators. Investigation of the DHP
binding domain within the
1A sequence background of
1ADHP not only reveals information about the molecular
mechanism of DHP interaction with L-type Ca2+ channel
1
subunits in general, but also provides structural hints for novel DHP
analogs that could possess considerable affinity for
1A subunits and
therefore represent lead compounds for further drug development.
We found that in the 1A sequence environment the two L-type
isoleucine residues in IIIS6 (Ile1504, Ile1507)
are not important for DHP interaction and can be replaced by the
respective
1A phenylalanines. Therefore transfer of high DHP
sensitivity to
1A requires the introduction of even less than 9 L-type residues as reported for
1ADHP (6, 7). From one
of our previous studies with
1A chimeras (9) it is evident that DHP
antagonist sensitivity only very weakly depends on the four L-type
residues in repeat IV (Fig. 1A). Therefore, DHP interaction with the remaining three L-type channel amino acid residues in IIIS5
and IIIS6 (Thr1393, Gln1397, and
Met1512) is crucial for DHP sensitivity of
1ADHP. The strongest structural determinant abolishing
DHP sensitivity in
1A is the tyrosine in position
Thr1393 which, according to our data, sterically prevents
DHP binding. In contrast, there is no evidence for such an effect for
1A residues introduced into positions 1397 and 1512. Instead, these
fail to provide suitable attachment sites for the DHP molecule thus
leading to a measurable but limited sensitivity decrease. It is
therefore likely that drug molecules (e.g. DHP derivatives)
that escape the steric hindrance of Tyr1393 and do not
depend on binding interaction with Met1512 and
Gln1397 must have considerable Ca2+ channel
blocking effects for
1A subunits. Smaller and more
hydrophobic compounds may fulfill these requirements.
Together with the results from earlier studies (9, 17, 18, 22, 29) our
data show that the structural requirements for effective stimulation by
DHP agonists of IBa through 1ADHP are more complex than for antagonists. This is based on the observation that introduction of the
1A sequence into several L-type positions of
1ADHP in repeats III (1393, 1397, 1512) and IVS6 (9)
results in a complete loss of agonist sensitivity to 10 µM concentrations of BayK8644 and FPL64176. Most of these
mutations and mutation Y1503A illustrate that DHP agonist effects can
be removed completely in mutants still displaying antagonist
sensitivity (e.g. Q1397M, Y1503A, M1512V; Fig. 3). In
radioligand binding studies DHP agonists and antagonists bind to their
binding domain in an apparently competitive manner. It is therefore
likely that such mutations still allow DHP agonist binding to the
channel but are unable to stabilize or prevent stabilization of open
channel conformations by the bound agonist. A similar conclusion was
reached after mutation of a pore-loop serine (position 1466 in
1ADHP), which prevents agonist effects but appears still
to mediate antagonist sensitivity (5).
In summary, our data provide important insight into DHP interaction
with their binding domain within the structural background of 1A.
This molecular information provides clues for the development of
1A-selective Ca2+ channel blockers.
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ACKNOWLEDGEMENTS |
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We thank Drs. T. Langer (Institute of Pharmaceutical Sciences), M. Grabner, S. Hering, and S. Berjukow for helpful discussions, Drs. M. Sinnegger and R. Kraus for reading the manuscript, and E. Emberger and D. Kandler for expert technical assistance.
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FOOTNOTES |
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* This work was supported by Austrian Science Fund Grants P12641 and P14820 (to J. S.) and P12689 (to H. G.), by the Österreichische Nationalbank, the Dr. Legerlotz Foundation, and European Community Research Training Network Grant HPRN-CT-2000 00082 (to J. 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.
To whom correspondence should be addressed. Tel.:
43-512-507-3164; Fax: 43-512-588-627; E-mail:
joerg.striessnig@uibk. ac.at.
Published, JBC Papers in Press, January 19, 2001, DOI 10.1074/jbc.M010164200
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ABBREVIATIONS |
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The abbreviations used are: DHP(s), 1,4-dihydropyridine(s); PCR, polymerase chain reaction; IBa, inward barium current(s).
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