Originally published In Press as doi:10.1074/jbc.M908836199 on April 13, 2000
J. Biol. Chem., Vol. 275, Issue 29, 22114-22120, July 21, 2000
Molecular Mechanism of Calcium Channel Block by Isradipine
ROLE OF A DRUG-INDUCED INACTIVATED CHANNEL CONFORMATION*
Stanislav
Berjukow,
Rainer
Marksteiner,
Franz
Gapp,
Martina J.
Sinnegger, and
Steffen
Hering
From the Institut für Biochemische Pharmakologie,
Peter-Mayr-Straße 1, A-6020 Innsbruck, Austria
Received for publication, November 1, 1999, and in revised form, April 12, 2000
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ABSTRACT |
The role of the inactivated channel conformation
in the molecular mechanism of Ca2+ channel block by
the 1,4-dihydropyridine (DHP) (+)-isradipine was analyzed in L-type
channel constructs (
1Lc; Berjukow, S., Gapp, F., Aczel,
S., Sinnegger, M. J., Mitterdorfer, J., Glossmann, H., and Hering,
S. (1999) J. Biol. Chem. 274, 6154-6160) and a DHP-sensitive class A Ca2+ channel mutant
(1A-DHP; Sinnegger, M. J., Wang, Z., Grabner, M.,
Hering, S., Striessnig, J., Glossmann, H., and Mitterdorfer, J. (1997)
J. Biol. Chem. 272, 27686-27693) carrying the high
affinity determinants of the DHP receptor site but inactivating at
different rates. Ca2+ channel inactivation was modulated by
coexpressing the 1A-DHP- or 1Lc-subunits
in Xenopus oocytes with either the 
2a- or
the 1a-subunit and amino acid substitutions in L-type
segment IVS6 (I1497A, I1498A, and V1504A). Contrary to a modulated
receptor mechanism assuming high affinity DHP binding to the
inactivated state we observed no clear correlation between steady state
inactivation and Ca2+ channel block by (+)-isradipine: (i)
a 3-fold larger fraction of 1A-DHP/1a
channels in steady state inactivation at 
80 mV (compared with
1A-DHP/2a) did not enhance the block by
(+)-isradipine; (ii) different steady state inactivation of
1Lc mutants at 30 mV did not correlate with
voltage-dependent channel block; and (iii) the
midpoint-voltages of the inactivation curves of slowly inactivating
L-type constructs and more rapidly inactivating
1Lc/1a channels were shifted to a
comparable extent to more hyperpolarized voltages. A kinetic analysis
of (+)-isradipine interaction with different L-type channel constructs
revealed a drug-induced inactivated state. Entry and recovery from
drug-induced inactivation are modulated by intrinsic inactivation
determinants, suggesting a synergism between intrinsic inactivation and
DHP block.
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INTRODUCTION |
Ca2+ channels are hetero-oligomeric protein complexes
consisting of a pore-forming 1-subunit, one out of at
least four -subunits (1-4), an
2/
-subunit, and in skeletal muscle an additional 
-subunit (3, 4). The auxiliary channel subunits, in particular the
-subunits, modulate voltage dependence, expression density, and
kinetics of Ca2+ channels (for review see Ref. 4). There is
biochemical evidence that multiple -subunits are associated with the
1-subunit, suggesting a tissue-specific modulation of
Ca2+ channel properties by different -subunit expression
patterns (5, 6).
The 1-subunits of L-type Ca2+ channels
(classes C (formed by 1C-subunits), D (formed by
1D-subunits), and S (formed by
1S-subunits)) possess drug receptors for
1,4-dihydropyridines (DHPs),1
phenylalkylamines, and benzothiazepines (7, 8). Mutational analysis of
L-type Ca2+ channel 1-subunits revealed nine
amino acid residues in segments IIIS5, IIIS6, and IVS6 that confer high
affinity and stereoselective interaction with DHPs (2, 9-13).
According to the modulated receptor hypothesis (14), the interaction of
an ion channel blocker with its receptor sites depends on whether the
channel is in a resting (closed), open (activated), or inactivated
(closed) conformational state. This hypothesis was applied to
Ca2+ channel block by organic calcium channel blockers such
as phenylalkylamines, benzothiazepines, and DHPs (15, 16). In the frame
of a specific version of the modulated receptor model, the more
efficient Ca2+ channel inhibition by DHPs at depolarized
membrane potentials is interpreted as predominant block of inactivated
channels. Accordingly, stronger DHP antagonist action in the
depolarized vascular tissue was suggested to reflect a more efficient
block of inactivated Ca2+ channels (but see Refs. 17 and
18).
To elucidate the role of the inactivated channel conformation in the
molecular mechanism of Ca2+ channel block by DHPs, we have
designed Ca2+ channels inactivating at different rates by
co-expressing a L-type channel construct 1Lc (1) or the
DHP-sensitive class A Ca2+ channel mutant
1A-DHP (2) with either 1a- or
2a-subunits. Additionally, Ca2+ channel
inactivation was modulated by introducing point mutations into segment
IVS6 of 1Lc. The consequences of changed inactivation properties for (+)-isradipine-induced block were analyzed by means of
the two-microelectrode voltage clamp technique after expression in
Xenopus oocytes. We observed no clear correlation between
the amount of steady state inactivation and
voltage-dependent block by (+)-isradipine. Our data support
a hypothesis where the inactivation gating of Ca2+ channels
is accelerated in their antagonist DHP-bound form. Channel constructs
with impaired fast inactivation displayed faster recovery from block by
(+)-isradipine, suggesting a close interdependence between intrinsic
inactivation determinants and the (+)-isradipine-induced inactivated state.
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EXPERIMENTAL PROCEDURES |
1 cDNAs--
Mutations I1497A, I1498A, and
V1504A were introduced into cDNA of the L-type channel construct
1Lc (1) by using an EcoRV-BstEII cassette (nucleotides 4542 and 4833). 1Lc is a construct
corresponding to rabbit 1C-a cDNA (19) with part of
the amino terminus replaced by carp 1S-sequence as
described (1, 10). Amino acid and nucleotide numbering of
Lc and mutants derived thereof is according to
1C-a cDNA sequence (19). All mutations were
introduced by polymerase chain reaction as described previously (10).
Fragments amplified by polymerase chain reaction were sequenced
entirely to confirm sequence integrity.
Electrophysiology--
Inward barium currents
(IBa) were studied with two microelectrode
voltage-clamp of Xenopus oocytes 2-7 days after
microinjection of approximately equimolar cRNA mixtures of
1Lc or 1A-DHP (2) (0.3 ng/50 nl) together
with 2 (0.2 ng/50 nl) and either 1a (0.1 ng/50 nl) or 2a (0.1 ng/50 nl) cRNA as described
previously (10). The corresponding constructs were named
1Lc/A-DHP/1a or
1Lc/A-DHP/2a channels. Mutant
1Lc-subunits (named herein I1497A, I1498A, and V1504A;
see above) were coexpressed with 2 and the
1a-subunit exclusively.
All experiments were carried out at room temperature in a bath solution
with the following composition: 40 mM Ba(OH)2,
50 mM NaOH, 5 mM HEPES, 2 mM CsOH
(pH adjusted to 7.4 with methanesulfonic acid). Voltage recording and
current injecting microelectrodes were filled with 2.8 M
CsCl, 0.2 M CsOH, 10 mM EGTA, 10 mM
HEPES (pH 7.4) and had resistances of 0.3-2 M
. Resting channel
block was estimated as peak IBa inhibition
during 100-ms test pulses from 80 to 20 mV at a frequency of 0.033 Hz
until steady state was reached. The dose response curves of
IBa inhibition were fitted using the Hill
equation:
IBa, drug/IBa, control (as percentages) = (100 A)/(1 + (C/IC50)nH) + A, where IC50 is the concentration at which
IBa inhibition is half-maximal, C is
the applied drug concentration, A is the fraction of
IBa that is not blocked, and
nH is the Hill coefficient.
Recovery from inactivation was studied at a holding potential of 80
mV after depolarizing Ca2+ channels during a 3-s prepulse
to 20 mV by applying 30-ms test pulses (to 20 mV) at various time
intervals after the conditioning prepulse. Peak
IBa values were normalized to the peak current measured during the prepulse, and the time course of
IBa recovery from inactivation was fitted to a
mono- or biexponential function (IBa, recovery = A × exp(t/
fast) + B × exp(t/slow) + C).
Voltage dependence of inactivation under quasi-steady state conditions
was measured using a multi step protocol to account for run-down (less
than 10%). A control test pulse (50 ms to 20 mV) was followed by a
1.5-s step to 100 mV followed by a 30-s conditioning step, a 4-ms
step to 100 mV, and a subsequent test pulse to 20 mV (corresponding
to the peak potential of the I-V curves).
Inactivation during the 30 s conditioning pulse was calculated as
follows.
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(Eq. 1)
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The pulse sequence was applied every 3 min from a holding
potential of 100 mV. Inactivation curves were drawn according to the
following Boltzmann equation.
![I<SUB><UP>Ba, inactivation</UP></SUB>=I<SUB><UP>SS</UP></SUB>+(1−I<SUB><UP>SS</UP></SUB>)(1+<UP>exp</UP>[(V−V<SUB>0.5</SUB>)/k)]](/content/vol275/issue29/fulltext/22114/img002.gif)
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(Eq. 2)
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where V is the membrane potential,
V0.5 is the midpoint voltage, k is
the slope factor, and Iss is the fraction of
noninactivating current.
Steady state inactivation of 1A-DHP and
1Lc channels at 80 mV was estimated by shifting the
membrane holding potential from 80 to 100 mV (1Lc)
or 120 mV (1A-DHP). Subsequent monitoring of the
corresponding changes in IBa amplitudes until
steady state revealed the fraction of Ca2+ channels in the
inactivated state at 80 mV. Steady state inactivation of different
L-type channel constructs at 30 mV was estimated by fitting time
course of current inactivation to a biexponential function (see Fig.
4A).
The IBa inactivation time constants were
estimated by fitting the IBa decay to a mono or
biexponential function. Data are given as the means ± S.E.
Statistical significance was calculated according to Student's
unpaired t test.
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RESULTS |
-Subunit Regulation of Ca2+ Channel Inactivation and
Block by (+)-Isradipine--
To evaluate the role of -subunit
mediated inactivation in Ca2+ channel block by isradipine,
we coexpressed the 1Lc (1) and 1A-DHP-subunits (2) together with either the
1a- or the 2a-subunit and analyzed peak
current inhibition by its high affinity (+)-enantiomer. In line with
previous observations (20), 1Lc/2a
channels displayed slower inactivation kinetics than
1Lc/1a channels (Fig.
1). At 80 mV
1Lc/1a channels displayed slightly more
steady state inactivation than 1Lc/2a
channels (p > 0.05; Fig. 1D). Slowly inactivating 1Lc/2a channels were blocked
by (+)-isradipine with a half-maximal inhibitory concentration of
198 ± 35 nM (n = 3), which was not
statistically different from block of more rapidly inactivating
1Lc/1a channels (IC50 = 327 ± 41 nM, p > 0.05; Fig.
1A).
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Fig. 1.
Inhibition of
1Lc/1a
and
1Lc/2a
channels by (+)-isradipine. A, concentration-response
relationships of peak IBa inhibition of
1Lc/1a ( ) and
1Lc/2a channels ( ) by (+)-isradipine.
Channel block was estimated as the ratio of peak current in the
presence of (+)-isradipine to that in control. Data points represent
the mean values from 4-8 experiments. The IC50 and the
Hill coefficients (nH) were obtained by best fit
of the data points to the general dose response equation (see
"Experimental Procedures") yielding:
1Lc/1a (): IC50 = 327 ± 41 nM, nH = 0.80 ± 0.06;
1Lc/2a (): IC50 = 198 ± 35 nM, nH = 0.87 ± 0.05. B,
representative scaled IBa of
1Lc/1a and
1Lc/2a channels during a 10-s
depolarizing step from 80 mV to 20 mV illustrate the different
inactivation properties. C, IBa decay
at the end of a 10 s depolarizing pulse (see B) of
1Lc/1a and
1Lc/2a channels as percentages.
D, fraction of 1Lc/1a and
1Lc/2a channels in steady state
inactivation at 80 mV as percentages.
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The DHP-sensitive class A Ca2+ channel mutant
1A-DHP inactivates at a faster rate than L-type channels
and displays an inactivation curve that is, compared with L-type
channels, significantly shifted into the hyperpolarizing direction (2).
The slightly higher DHP sensitivity of this channel construct compared
with L-type channels could therefore reflect a more efficient drug
interaction with the inactivated channel state at 80 mV.
To test this hypothesis we coexpressed the
1A-DHP-subunit with different -subunits and analyzed
the (+)-isradipine sensitivity of the two channel constructs
inactivating at different rates. As shown for
1Lc/2a (Fig. 1B), coexpression
of the 2a-subunit dramatically slowed the inactivation
time course of the resulting 1A-DHP/2a
channels (Fig. 2B).
Furthermore, at 80 mV we observed a 3-fold larger steady state
fraction of 1A-DHP/1a channels in
inactivation (46 ± 2%, n = 4) than in
1A-DHP/2a (16 ± 2%, n = 5, p < 0.01; Fig. 2D).
A modulated receptor mechanism implying high affinity block of
inactivated channels would predict a more pronounced inhibition of
1A-DHP/1a channels. The concentration of
(+)-isradipine for half-maximal block of IBa in
1A-DHP/1a channels (IC50 = 61 ± 14, n = 4) at 80 mV was, however, not
significantly different from 1A-DHP/2a
(IC50 = 52 ± 14, p > 0.05; Fig.
2A). These data clearly indicate that enhanced inactivation
did not cause stronger IBa inhibition of
1A-DHP/1a channels.
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Fig. 2.
Inhibition of
1A-DHP/1a
and
1A-DHP/2a
channels by (+)-isradipine. A, concentration-response
relationships of peak IBa inhibition of
1A-DHP/1a and
1A-DHP/2a channels by (+)-isradipine.
Data points were fitted to the general dose response equation (as shown
under "Experimental Procedures"):
1A-DHP/1a ( ): IC50 = 61 ± 14 nM, nH = 0.97 ± 0.05;
1A-DHP/2a ( ): IC50 = 52 ± 14 nM, nH = 0.87 ± 0.05 (n  3). B, representative scaled
IBa of 1A-DHP/1a
and 1A-DHP/2a channels during a 3-s
depolarizing step from 80 mV to 20 mV. C,
IBa decay of
1A-DHP/1a and
1A-DHP/2a channels at the end of a 3 s pulse (see B) as percentages. D, fraction of
1A-DHP/1a and
1A-DHP/2a channels in steady state
inactivation at 80 mV as percentages. *, statistically significant
different with p < 0.01.
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Inactivation Determinants in Segment IVS6 Affect Ca2+
Channel Block by (+)-Isradipine--
To evaluate the role of channel
inactivation determinants in the pore forming
1Lc-subunit for channel block by (+)-isradipine, we
replaced three amino acids in segment IVS6 of 1Lc by
alanine (I1497A, I1498A, and V1504A) and analyzed the DHP sensitivity of the resulting mutants.
As shown in Fig. 3A, alanine
substitutions of the two putative DHP-binding determinants (I1497A and
I1498A; Ref. 7) and mutation V1504A (1) significantly slowed channel
inactivation kinetics. None of the point mutations reduced
Ca2+ channel inhibition by (+)-isradipine (Fig.
3B). Instead, we observed a significant decrease in the
half-maximal inhibitory concentrations for peak
IBa inhibition of I1497A (IC50 = 28 ± 10 nM), I1498A (IC50 = 50 ± 11 nM), and V1504A (IC50 = 71 ± 15 nM) compared with 1Lc/1a
(IC50 = 327 ± 41 nM, p < 0.05, n 4; Fig. 1).
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Fig. 3.
Inactivation determinants in
L-type segment IVS6 and Ca2+ channel block by
(+)-isradipine. A, comparison of
IBa inactivation of
1Lc/1a channels with mutants I1497A,
I1498A, V1504A, and 1Lc/2a channels
measured as IBa decay during a 3-s test pulse
from 80 mV to 20 mV. All constructs displayed statistically
significant slower current inactivation compared with
1Lc/1a channels (p < 0.01, n = 5-12). Inset, representative
IBa of the indicated Ca2+ channel
constructs were scaled to illustrate the different inactivation time
courses. B, concentration-response relationship of peak
IBa inhibition by (+)-isradipine. Data points
represent the mean values from 3-8 experiments. Fitting the data
points to the general dose response equation yielded: I1497A ():
IC50 = 28 ± 10 nM, nH = 0.81 ± 0.04; I1498A ( ): IC50 = 50 ± 11 nM, nH = 1.17 ± 0.07; I1504A ():
IC50 = 71 ± 15 nM, nH = 1.35 ± 0.06. For IC50 and nH of
1Lc/1a (, dashed line)
see Fig. 1.
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At a holding potential of 80 mV, the fraction of inactivated channels
was not significantly different between the various L-type channel
constructs (ranging from 5 ± 2% (V1504A, n = 5) to 3 ± 1% (1Lc/1a,
n = 5)). The observed changes in drug sensitivity at
80 mV appear, therefore, to be associated with a more pronounced resting channel block.
Steady state Inactivation and Voltage-dependent L-type
Ca2+ Channel Block--
To evaluate the role of the
inactivated channel conformation of our L-type constructs as high
affinity receptor for (+)-isradipine, we estimated the steady state
inactivation at 30 mV (see pulse protocol in inset of Fig.
4A). Different channel
constructs displayed significantly different steady state inactivation
at 30 mV (ranging in the absence of drug between 9 ± 1%
(1Lc/1a, n = 5) and
21 ± 3% (I1498A, n = 4)) (Fig. 4, C
and D).
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Fig. 4.
Isradipine-induced inactivation in resting
Ca2+ channels. A and B, effect
of prepulses of variable duration (inset) to a subthreshold
voltage of 30 mV on the current evoked by a test pulse to 20 mV
(holding potential, 80 mV). The currents were normalized to that
observed during a test pulse to 20 mV given alone (no prepulse
applied). The smooth curves are biexponential functions fitted to the
time course of mean IBa inactivation (± S.E.)
of 1Lc/1a (n = 4, A) and of 1Lc/2a channels
(n = 4, B) in control (), the presence of
(+)-isradipine () (1 µM). The parameters of the fit to
time-dependent IBa inactivation
during conditioning pulses of different length
IBa/normalized = Afast × exp(t/fast) + Aslow × exp(t/slow) + C were in
A (1Lc/1a) ():
Afast = 0.04, fast = 0.65 s,
Aslow = 0.05, slow = 14.0 s,
C = 0.91; (): Afast = 0.39, fast = 0.57 s, Aslow = 0.23, slow = 14 s, C = 0.37; in
B (1Lc/2a) ():
Afast = 0.06, fast = 0.92 s,
Aslow = 0.12, slow = 11.4 s,
C = 0.83; (): Afast = 0.28, fast = 0.69 s, Aslow = 0.25, slow = 12.9 s, C = 0.45. The
dashed lines illustrate the steady state inactivation in
control and the presence of 1 µM isradipine. The
arrows in A and B illustrate the
additional drug-induced steady state inactivation. C,
additional steady state inactivation in the indicated channel
constructs induced by 1 µM (+)-isradipine. Drug-induced
fast inactivation (Afast, drug Afast, control) is illustrated by the
black columns. D, steady state inactivation in
control plotted versus steady state inactivation in
(+)-isradipine at 30 mV.
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We did, however, not observe a simple relation between inactivation and
voltage-dependent channel block by (+)-isradipine. As
illustrated in Fig. 4 (A and B), a higher
fraction of steady state inactivation in
1Lc/2a channels did not result in a
stronger voltage-dependent channel block compared with
1Lc/1a channels. Compared with its effect
on 1Lc/1a channels (56 ± 3%
additional current inhibition, n = 4), the drug induced
significantly less channel block in
1Lc/2a (40 ± 4%), I1498A (25 ± 2%), and V1504A channels (34 ± 5%, n > 7;
Fig. 4C). Plotting the fraction of steady state inactivation
of the different L-type channel mutants at 30 mV against the amount
of drug-induced inactivation revealed even a reversed correlation
(r = 0.94) between these two parameters (Fig.
4D).
The different inactivation properties of constructs
1Lc/1a,
1Lc/2a, I1498A, and V1504A prompted us to
analyze the voltage dependence of channel inactivation in control and
the presence of (+)-isradipine in more detail. The inactivation curves
(measured with 30 s conditioning prepulses) in control and the
presence of 10 nM, 100 nM, and 1 µM (+)-isradipine are illustrated in Fig. 5. The midpoint voltages of the
inactivation curves of all four L-type constructs were shifted to a
comparable extent in the hyperpolarizing direction (Fig. 5 and Table
I). A modulated receptor mechanism would
predict a correlation between channel inactivation during the
conditioning prepulses and voltage-dependent channel block. No such correlation was observed.
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Fig. 5.
Modulation of the inactivation curves of
1Lc/1a,
I1497A, I1498A, V1504A, and
1Lc/2a
channels by (+)-isradipine. Voltage dependence of Ca2+
channel inactivation during 30-s depolarizing test pulses. Data point
were fitted to a Boltzmann function (see "Experimental
Procedures"). The mean values for corresponding slope parameters
(k) and half-maximal inactivation potential
(V0.5) in control (), 10 nM
(), 100 nM (), and 1 µM ()
(+)-isradipine are shown in Table I.
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Table I
Slope factors (k) and midpoint voltages (V0.5) of the
inactivation curves shown in Fig. 5
Corresponding shifts of V0.5 in the presence of 1 µM: 24 ± 3 (1Lc/1a), 21 ± 4 (1Lc/2a), 21 ± 3 (I1498A), and 19 ± 3 (V1504A) were not significantly different (p > 0.1).
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Kinetics of Drug-induced Inactivation at 30
mV--
Ca2+ channel inactivation at 30 mV developed
with a biexponential time course (Fig. 4). The fast inactivation time
constant (fast) ranged between 0.42 ± 0.06 s
(I1497A) and 0.92 ± 0.07 s
(1Lc/2a), and the slow inactivation time
constant (slow) ranged between 10.1 ± 2.1 s
(I1497A) and 14.0 ± 2.1 s
(1Lc/1a). (+)-Isradipine (1 µM) induced an additional fast component in Ca2+ channel inactivation developing with similar kinetics
as fast inactivation in control. The fast inactivation time constant
(fast, isradipine) ranged in the different constructs
between 0.51 ± 0.06 s (I1498A) and 0.89 ± 0.08 s
(I1497A). The slow component in channel inactivation at 30 mV was not
significantly affected by the drug (Fig. 4, A and
B). These findings suggest that (+)-isradipine promotes a
channel conformation resembling intrinsic fast inactivation. The
fraction of channels in this new drug-induced inactivated state
correlated with the drug-induced steady state inactivation (Fig.
4C).
(+)-Isradipine-induced Changes in IBa Kinetics at 20 mV--
(+)-Isradipine-induced changes in IBa
kinetics at 20 mV are illustrated in Fig.
6. The drug (1 µM)
accelerated the fast component in IBa decay of
1Lc/1a from fast, control = 415 ± 52 ms to fast, isradipine = 166 ± 9 ms (n = 7). In 1Lc/2a and
V1504A channels IBa inactivation developed with
slow time constants of 16 ± 2 s
(1Lc/2a) and 12 ± 2 s (V1504A,
n = 5), indicating complete absence of intrinsic fast
inactivation (see also corresponding recovery experiments in Fig.
7). (+)-Isradipine induced in both
channel constructs a transient component in the current decay with
similar time constants of
fast, isradipine(V1504A) = 215 ± 35 ms
and fast, isradipine
(1Lc/2a) = 210 ± 30 ms
(n = 5). The slow inactivation time constants of the
IBa decay were not significantly affected by the
drug (see right column of Fig. 6 and inset in
Fig. 6A).
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Fig. 6.
Modulation of current decay by
(+)-isradipine. Representative IBa through
the indicated channel constructs during 3-s depolarizations from 80
to 20 mV in control () and the presence of 1 µM
(+)-isradipine () (left panels).
IBa were scaled to illustrate
(+)-isradipine-induced changes in the current decay. The
inset in A illustrates representative
IBa of 1Lc/1a
channels during 30-s depolarizations under the same conditions. The
time constants of fast (fast, middle panels)
and slow (slow, right panels)
IBa decay in control (white columns)
and 1 µM (+)-isradipine (black columns) were
estimated by fitting biexponential functions to the current traces
evoked by 30-s depolarizing pulses from 80 to 20 mV. Data from 6-12
experiments are shown. No fast component in inactivation was observed
for 1Lc/2a and V1504A. In
1Lc/1a the relative amplitudes changed
from Afast, control = 0.50 ± 0.08 to
Afast, drug = 0.75 ± 0.03 and in and
I1498A from Afast, control = 0.11 ± 0.03 to Afast, drug = 0.41 ± 0.07.
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Fig. 7.
Recovery from isradipine-induced
inactivation. Time course of IBa recovery
from inactivation, after a 3-s conditioning prepulse to 20 mV (holding
potential, 80 mV). Test pulses to 20 mV were applied at various time
intervals after the conditioning pulses. Peak
IBa during the test pulses were normalized to
peak IBa measured during the conditioning
prepulse. Smooth curves in A-D represent mono-
or biexponential functions fitted to IBa
recovery (means ± S.E., n = 4-7) of
1Lc/1, I1498A,
1Lc/2, and V1504A channels in control
() and in 1 µM (+)-isradipine (). The parameters of
fit were as follows. A, (): A = 0.32, = 0.63 s, C = 1; ():
A = 0.68, = 2.85 s, C = 0.98; B, (): Afast = 0.13, fast = 0.50 s, Aslow = 0.21, slow = 12 s, C = 0.99; ():
Afast = 0.46, fast = 0.47 s,
Aslow = 0.20, slow = 12 s,
C = 0.97; C, (): A = 0.09, = 6.1 s, C = 0.99; (): A = 0.42, = 2.26 s, C = 0.98; D,
(): A = 0.10, = 11.7 s,
C = 0.98; (): Afast = 0.36, fast = 1.48 s, Aslow = 0.20, slow = 14.5 s, C = 0.98. E, time constants of recovery from fast inactivation in
control (white columns) and 1 µM
(+)-isradipine (black columns).
1Lc/2a, I1498A, and V1504A channels
recover at a significantly faster rate from (+)-isradipine-induced
inactivation than 1Lc/1 channels (# with
p < 0.01). F, drug-induced fast
inactivation (Afast, drug Afast, control) determined from recovery
experiments (see A-D). The fraction of drug-inactivated
channels was neither affected by single point mutations in segment IVS6
nor by expression of different -subunits.
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Mutation I1498A reduced Ca2+ channel inactivation (Fig.
3A) and simultaneously accelerated fast inactivation
(fast, control = 155 ± 20 ms,
p < 0.01 compared with 415 ± 52 1Lc/1a; see also Fig. 6C).
Irrespective of the changes in channel inactivation in I1498A channels,
drug-induced acceleration of the current occurred at a similar rate as
in 1Lc/1a channels
(fast, isradipine = 142 ± 18 ms,
n = 6, Fig. 6, A and C). Thus,
(+)-isradipine-induced acceleration of the current decay occurred in
all L-type channel constructs at a comparable rate.
Recovery from Voltage-dependent Block by
(+)-Isradipine--
IBa recovery of
1Lc/1a channels at 80 mV was well
described by a monoexponential function (rec, control = 0.65 ± 0.07 s, n = 6; Fig. 7A).
Recovery in the presence of 1 µM (+)-isradipine was about
four times slower (rec, drug = 2.80 ± 0.14 s, n = 4; Fig. 7, A and E),
suggesting that the drug promotes a new (drug-induced) inactivated state.
Regardless of the complete absence of intrinsic "fast inactivation"
in 1Lc/2a and V1504A channels (Fig. 6,
B and D), (+)-isradipine (1 µM)
induced a transient component in IBa decay and a
corresponding fast component in recovery with kinetics comparable with
drug-modulated 1Lc/1a channels
(rec, drug [1Lc/2a] = 2.26 ± 0.26 s, n = 3 and
rec, drug [V1504A] = 1.48 ± 0.48 s,
n = 5; Fig. 7, C-E).
Mutant I1498A recovered with biexponential kinetics in control (Fig.
7B). (+)-Isradipine enhanced the impact of the fast
component (Fig. 7F). However, the drug did not slow the time
course of IBa recovery in I1498A channels.
Neither the recovery time constant from fast inactivation nor recovery
from slow inactivation were significantly affected by
(+)-isradipine (rec, control = 471 ± 50 ms,
rec, drug = 453 ± 45 ms, n = 4;
see also Fig. 7, B and E). Recovery of I1498A
channels from inactivation in the presence of (+)-isradipine occurred
at a 6-fold faster rate the recovery of
1Lc/1a channels, suggesting a
particularly important role of Ile1498 in stabilization of
the DHP-induced inactivated state.
| |
DISCUSSION |
According to a widely accepted hypothesis, DHPs bind with high
affinity to the inactivated state of Ca2+ channels (15,
16). We have revisited this concept and analyzed the (+)-isradipine
sensitivity of several L-type and DHP-sensitive class A
Ca2+ channel constructs with different inactivation properties.
Resting State Block by (+)-Isradipine Is Affected by Mutations in
L-type Segment IVS6--
Our data clearly demonstrate that two
putative DHP binding determinants in segment IVS6 (Ile1497
and Ile1498; Ref. 11) form part of the L-type channel
inactivation mechanism (Fig. 3A). Alanine substitutions of
these amino acids slowed IBa inactivation and
increased the apparent DHP sensitivity at 80 mV (Fig. 3). Slowing the
kinetics of 1Lc channels by coexpression of the
2a-subunit had no significant effect on
IBa inhibition (Figs. 1 and 3). However, neither
co-expression of the different 1Lc mutants with the
2a-subunit nor introducing point mutations into segment
IVS6 significantly affected the steady state fraction of channels in
the inactivated state at 80 mV. Our measurements of drug action at
80 mV therefore primarily provided information about an enhanced
resting state affinity of constructs I1497A, I1498A, and V1504A.
Role of Inactivation in 1A-DHP Channel Block by
(+)-Isradipine--
As illustrated in Fig. 2, we estimated at 80 mV
an about 3-fold larger fraction of
1A-DHP/1a channels in steady state
inactivation than in 1A-DHP/2a. Despite
this marked difference, 1A-DHP/1a and
1A-DHP/2a channels were blocked with
similar IC50 values (1A-DHP/1a, 61 ± 14;
1A-DHP/2a, 52 ± 14;
p > 0.05).
This result is hard to explain in the frame of a modulated receptor
model suggesting high affinity drug binding to the inactivated channel
conformation. Instead, our data clearly demonstrate that -subunit-induced changes in the steady state fraction of channel inactivation at 80 mV have no significant effect on (+)-isradipine sensitivity of 1A-DHP channels. A higher sensitivity of
mutant 1A-DHP/1a (IC50 = 61 ± 14) compared with the L-type construct 1Lc/1a (IC50 = 327 ± 41, p < 0.01) therefore cannot be explained by a high
affinity block of a larger fraction of 1A-DHP channels in an inactivated state. A loss in voltage dependence of DHP block caused by the class A sequence environment of the DHP-binding site in
1A-DHP cannot be excluded.
Role of Inactivation in L-type Ca2+ Channel Block by
(+)-Isradipine--
Next we exploited the different inactivation
properties of our L-type Ca2+ channel constructs to analyze
whether a larger steady state fraction of inactivated channels at 30
mV would enhance voltage-dependent channel block. As shown
in Fig. 4D, we observed even a reversed correlation between
these two parameters.
This apparent discrepancy with a high affinity drug binding mechanism
to the inactivated channel state is also illustrated in Fig. 5. Removal
of fast inactivation in V1504A and in
1Lc/2a channels substantially reduced the
fraction of inactivated channels during the 30-s conditioning pulses
compared with 1Lc/1a (Fig. 5,
A-C). We observed, however, no correlation between the
shifts in the midpoint voltages of the inactivation curves in the
presence of 10 nM to 1 µM (+)-isradipine
(Table I) and the amount of inactivation during a 30-s conditioning
test pulse (more than 90% in 1Lc/1a channels versus about 70% in
1Lc/2a and V1504A channels). These data
suggest that drug binding to the inactivated state is less crucial for
L-type channel block than previously supposed.
The kinetics of the voltage-dependent channel block by
(+)-isradipine at 30 mV support, however, the hypothesis that the drug promotes a conformational state resembling intrinsic fast inactivation (Fig. 4, A and B). As demonstrated
in Fig. 4, the formation of this "drug-induced inactivated state"
was modulated by intrinsic determinants of fast inactivation in segment
IVS6 and 1--subunit interaction.
At 20 mV (+)-isradipine (1 µM) accelerated the
IBa decay of all L-type channel constructs to a
comparable extent (Fig. 6). At this potential drug-induced acceleration
of the current decay could be due to rapid open channel block (21),
drug-induced inactivation, or both. It is tempting to speculate that
the (+)-isradipine-induced acceleration of the current decay at 20 mV
at least partially reflects drug-induced transitions of open channels
to an inactivated state that was observed for resting channels at 30
mV (Fig. 4, A and B). However, state transitions
of resting channels at 30 mV cannot be extrapolated to 20 mV, where
inactivation occurs predominantly from the open state. More detailed
studies including an analysis of the dose dependence of
(+)-isradipine-induced changes in IBa kinetics
at different membrane voltages are required to answer this question.
Intrinsic Inactivation Determinants in Segment IVS6 Affect Recovery
from (+)-Isradipine Block--
As shown in Fig. 7, constructs
1Lc/2a (rec, drug = 2.26 ± 0.23 s, n = 5) and V1405A
(rec, drug = 1.48 ± 0.48 s,
n = 4) recovered from the drug-induced inactivation
with slightly faster time constants than
1Lc/1a (rec, drug = 2.80 ± 0.14 s, n = 4, p = 0.05). Most dramatic effects were observed for amino acid substitution
I1498A. This mutant recovered in (+)-isradipine much faster from
inactivation (rec, drug = 0.45 ± 0.05 s,
n = 4) than 1Lc/1a and
the other constructs (Fig. 7E). This property distinguishes
Ile1498 from other IVS6 residues investigated in the
present study (Fig. 7B). A simple interpretation of these
results is that mutation I1498A and to a lesser extent mutation V1405A
and the 1Lc-2a interaction destabilize
the drug-induced inactivated state (Fig. 8).
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|
Fig. 8.
Intrinsic inactivation determinants affect
the stability of the (+)-isradipine-induced channel conformation.
Slower IBa recovery of L-type Ca2+
channels in the presence of (+)-isradipine suggests that the drug
stabilizes a new inactivated channel conformation (Fig. 7A;
see also Ref. 26). The scheme illustrates an interdependence between
intrinsic inactivation properties and the stability of the
(+)-isradipine-induced conformation. Amino acid substitution I1498A
induced a 6-fold faster IBa recovery from the
drug-induced state, and an about 2-fold faster recovery was observed
for mutant V1504A. Faster recovery of
1Lc/2a channels was significant (compared
with 1Lc/1a) but less pronounced (Fig. 7;
see also Ref. 26).
|
|
Implications for the Molecular Mechanism of DHP Action--
A role
of channel inactivation determinants in different sequence stretches of
L-type and non-L-type 1-subunits in block by DHPs was
earlier reported by Zuhlke et al. (22), Lacinova et
al. (23), and Bodi et al. (24).
An interesting question about the interaction of DHPs with
Ca2+ channels is whether these drugs bind with high
affinity to the inactivated state or whether the inactivation gating of
the channels in their drug-bound form is changed. In his original work
on the modulated receptor hypothesis, Hille (25) stressed both ways of
expressing the concept of the modulated receptor hypothesis.
The aim of the present study was to distinguish the impact of drug
binding to the inactivated state from drug-induced inactivation by
making use of DHP-sensitive Ca2+ channel constructs with
different inactivation properties. We report here almost identical
(+)-isradipine sensitivity of 1A-DHP/1a and 1A-DHP/2a channels despite the
pronounced differences in steady state inactivation (Fig. 2). For our
L-type mutants, we observed at 30 mV even a reversed correlation
between the different channel fractions in inactivation and
voltage-dependent channel block (Fig. 4D). Both
findings are inconsistent with a modulated receptor mechanism implying
an interdependence between high affinity DHP-binding to
Ca2+ channels in an inactivated state and drug-induced inactivation.
We hypothesize that drug-induced changes in the availability curve
reflect the formation of a new (+)-isradipine-induced inactivated channel conformation. Recovery from drug-induced inactivation is
modulated by intrinsic inactivation determinants, suggesting a
synergism between both processes (most prominent for mutation I1498A).
The possible structural implications of our data are hard to
interpret. On one hand, this interrelationship may indicate that conformational changes during channel inactivation modulate the orientation of DHP-binding determinants; on the other hand, they may also reflect an overlap between drug binding and inactivation determinants (see Fig. 8 for illustration).
In conclusion, DHP-sensitive Ca2+ channel constructs with
different inactivation properties represent valuable tools for studying the role of the inactivated channel conformation in DHP block. The
characterization of the drug-induced inactivated conformational state(s) and the identification of further structural links between the
DHP-binding sites and intrinsic channel inactivation is an exciting
subject for future mutational studies.
| |
ACKNOWLEDGEMENTS |
We thank Prof. H. Glossmann for providing the
cDNA of the class A mutant 1A-DHP and Dr. E. N. Timin, D. J. Beech, and S. Sokolov for comments on the manuscript.
We thank Dr. A. Schwartz for providing the 1C-a and
2/ cDNA and B. Kurka and E. Markreiter for expert
technical assistance.
| |
FOOTNOTES |
*
This work was supported by Fonds zur Förderung der
Wissenschaftlichen Forschung Grants 12649-MED (to S. H.) and Grant
12828-MED (to S. H.), a grant of the Else-Kröner-Fresenius
Stiftung, and a grant from the Austrian National Bank (to S. H.).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.: 43-512-507-3154;
Fax: 43-512-588627; E-mail: Steffen.Hering.@uibk.ac.at.
Published, JBC Papers in Press, April 13, 2000, DOI 10.1074/jbc.M908836199
| |
ABBREVIATIONS |
The abbreviation used is:
DHP, dihydropyridine.
| |
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TOP
ABSTRACT
INTRODUCTION
