 |
INTRODUCTION |
Voltage-gated Ca2+ channels mediate Ca2+
currents in muscle, endocrine cells, and neurons. They regulate a
variety of important cell functions such as contractility,
excitability, secretion, and gene expression. A distinct feature of
L-type Ca2+ channels is their high affinity for
Ca2+ antagonists (1). The stereoselective, high affinity
drug receptors for Ca2+ antagonists such as
1,4-dihydropyridines, phenylalkylamines
(PAAs),1 and benzothiazepines
(BTZs) are located on the pore-forming
1 subunit of
L-type channels (classes C, D, and S) (for review see Refs.
2 and 3).
Recent studies on cloned Ca2+ channel
1
subunits provided first insights into the molecular architecture of the
drug binding domains for Ca2+ antagonists. In a "loss of
function" approach receptor determinants of Ca2+
antagonists were identified by replacing putative drug binding sequences by corresponding sequence stretches of non-L-type
channels or by systematic alanine scanning mutagenesis (4-7).
Alternatively, in a "gain of function" approach,
1,4-dihydropyridine, PAA, and BTZ sensitivity could be transferred to
1A or
1E subunits by exchanging portions
of non-L-type sequences or even single amino acid residues
with the corresponding L-type counterparts (8-12). The
crucial structural elements of the Ca2+ antagonist-binding
domains involve amino acids in transmembrane segments IIIS5, IIIS6, and
IVS6. Adjacent IIIS5-IIIS6 and IVS5-IVS6 linkers contain additional,
less crucial drug binding determinants (see Ref. 3 for review).
We have previously demonstrated that transfer of the three high
affinity determinants of the PAA receptor (Tyr1804,
Ala1808, and Ile1811; see Ref. 11) to segment
IVS6 of the
1A subunit enhances the BTZ sensitivity of
the resulting class A channel mutant (Ref. 12; see also Ref. 13).
Recent photoaffinity labeling experiments by Kraus et
al. (14) suggest additional BTZ receptor determinants in the
segment IIIS6 of L-type channels (see also Ref. 15).
Here, we compare the PAA and BTZ sensitivity of two class
A/L-type Ca2+ channel chimeras consisting of
L-type sequence in transmembrane segments IIIS6 and IVS6
and adjacent S5-S6 linkers that was derived from either carp skeletal
muscle
1S (chimera AL1) or the rabbit heart
1C-a (AL16) and introduced into the sequence background of
1A (Fig. 1A). From previous work (12-14,
16) chimeras AL1 (8) and AL16 (12, 17) were expected to carry the
determinants for high affinity BTZ binding in their L-type
segments IIIS6 and IVS6. Surprisingly, only the chimeric channel
containing sequence stretches of the cardiac
1C-a
subunit was efficiently blocked by (+)-cis-diltiazem. Sequence
comparison of
1S and
1C-a revealed two
nonconserved amino acids in segment IVS6. Transfer of a single amino
acid (Ile1487) from segment IVS6 of the cardiac
1C-a sequence to the carp skeletal muscle
1S enhanced BTZ sensitivity of the resulting mutant to
the level of the
1A/
1C-a chimera. The
second divergent amino acid (Val1504,
1C-a
numbering) was identified as a strong class C channel inactivation determinant.
BTZ blocks Ca2+ channels in a use-dependent
manner (18). The structural basis of state-dependent
Ca2+ channel block by BTZ is not sufficiently understood.
Use-dependent block can be attributed to
state-dependent removal of guarding structures or high
affinity drug binding to open or inactivated channels during membrane
depolarization (19, 20).
A detailed analysis of the impact of both amino acids for
Ca2+ channel block of
1C-a mutants enabled
not only the identification of additional determinants of the diltiazem
receptor site but also provided new insights into the molecular
mechanism of L-type Ca2+ channel block by BTZ.
Our data suggest that besides the putatively pore orientated amino
acids in the central part of segment IVS6 (12), the BTZ receptor
contains an additional receptor determinant that is located near the
extracellular channel mouth. Furthermore, a residue in segment IVS6
(Val1504) that is localized close to the intracellular
mouth of the pore and determines inactivation also appears to modulate
drug binding. We obtained first structural evidence for a guarded and
modulated BTZ receptor of L-type channels.
 |
EXPERIMENTAL PROCEDURES |
Chimeric Class A/L-type Channel
Constructs--
Chimeras AL1 and AL16 were generated by replacing the
transmembrane segments IIIS6 and IVS6 and the adjacent S5-S6 linkers of
the rabbit brain class A Ca2+ channel (BI-2)
1A (21) by corresponding sequences of either the carp
skeletal muscle
1S (AL1; see Ref. 8) or the rabbit cardiac
1C-a (AL16; see Ref. 17).
Mutant
1 Subunits--
Mutant L1383I (amino acid
numbering according to
1S; see Ref. 22) was constructed
by introducing point mutations into
1S-cDNA (8).
cDNAs were inserted into a
KpnI*-BglII-cassette of chimera AL1
(nucleotides 5467 and 6185, numbering according to
1A).
The KpnI* site was generated by introducing a
silent mutation. I1487L, I1487A, and V1504A were introduced into
1C-a-cDNA of the L-type construct Lc
using a EcoRV-BstEII-cassette (nucleotides 4542 and 4833). Lc is a construct corresponding to
1C-a-cDNA (23) with part of the amino terminus
replaced by carp
1S sequence (to increase expression
density; see Ref. 8) containing two additional restriction sites
(HindIII/2955; SalI/3670). Amino acid and
nucleotide numbering is according to
1C-a (see Ref.
23).
All mutations were introduced by using polymerase chain reaction, which
was performed with proof-reading Pfu polymerase
(Stratagene). Fragments amplified by polymerase chain reaction were
sequenced entirely to confirm sequence integrity.
Electrophysiology--
Two microelectrode voltage-clamp of
Xenopus oocytes was performed 2-7 days after microinjection
of cRNAs in approximately equimolar mixtures of
1 (0.3 ng/50 nl)/
1a (0.1 ng/50 nl)/
2
(0.2 ng/50 nl) as described previously (8). All experiments were carried out
at room temperature in bath solution with the following composition: 40 mM Ba(OH)2, 40 mM
N-methyl-D-glucamine, 10 mM HEPES,
10 mM glucose (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
. Activation of endogenous Ca2+-activated Cl
conductance by barium
influx through Ca2+ channels was eliminated by injecting
50-100 nl of a 0.1 M
1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid
solution (adjusted with methanesulfonic acid to pH 7.0) into oocytes
20-40 min before the experiments. The recording chamber (150 µl of
total volume) was continuously perfused at a flow rate of 1 ml/min with
control or drug-containing solutions. The pClamp software package
(version 6.0, Axon Instruments, Inc.) was used for data acquisition and
analysis. Data were digitized at 2 kHz, filtered at 1 kHz, and stored
on a computer hard disk. Leakage current correction was performed by
using average values of scaled leakage currents elicited by a 10 mV
hyperpolarizing voltage step.
Tonic ("resting state-dependent") block was measured as
peak IBa inhibition during the first pulse after a 3-min
equilibration in drug-containing solution at rest (holding potential
80 mV), and use-dependent current inhibition was
subsequently estimated during 0.1 or 1 Hz trains of 100-ms test pulses.
Inactivation of IBa during a pulse train was estimated by
applying similar test pulses in the absence of drug.
Recovery of IBa from inactivation was studied at
80 mV by
depolarizing Ca2+channels during a 3-s prepulse to 20 mV
and subsequent application of a second test pulse to 20 mV at various
time intervals after the conditioning prepulse. Peak IBa
values were normalized to the peak current measured during the
prepulse. IBa recovered between 90 and 100% during a
subsequent 3-min rest at
100 mV. The time course of IBa
recovery from inactivation was fit to single or biexponential functions
(IBa, recovery = A*exp(
t/
rec, fast) + B*exp(
t/
rec, slow) + C). Data are given as the means ± S.E. Statistical
significance of IBa block by diltiazem or gallopamil compared with current decay under control conditions was
calculated according to unpaired Student t test
(p < 0.05).
 |
RESULTS |
Newly Identified Determinants of Ca2+ Channel Block by
(+)-cis-Diltiazem in Segment IVS6--
Fig.
1A shows a schematic
representation of the class A/L-type chimeras AL1 and AL16
consisting of either L-type sequence of carp skeletal
muscle
1S (AL1) or rabbit heart
1C-a
(AL16) in segments IIIS6 and IVS6 and adjacent S5-S6 loops. Previously identified determinants of the BTZ receptor site in the central part of
the putative pore-lining
-helical IVS6 segment (Tyr1386,
Ala1390, and Ile1393; see Refs. 12 and 13) are
highlighted in Fig. 1B. The amino acid sequences of
1C-a and
1S IVS6 segments are identical
with the exception of two nonconserved amino acid residues (Fig.
1C). Leu1383
Ile and Ile1400
Val represent conservative substitutions as each of the residues is
nonpolar, hydrophobic, and of comparable size. As shown in Fig.
1D, corresponding IBa values of chimeras AL1 and
AL16 displayed different kinetic properties with chimera AL1
inactivating significantly faster (52 ± 1% IBa
inactivation during a 300-ms pulse from
80 to 20 mV,
n = 18) than AL16 (31 ± 2%, n = 13, p < 0.001) (Fig. 1D).

View larger version (32K):
[in this window]
[in a new window]
|
Fig. 1.
Schematic representation of class
A/L-type Ca2+ channel chimeras AL1 and
AL16. A, class A ( 1A) sequence is shown
as white transmembrane segments and L-type
sequences from carp skeletal muscle ( 1S, AL1) or rabbit
heart ( 1C-a, AL16) in segments IIIS6 and IVS6, and
adjacent S5-S6 connecting loops are illustrated as black
segments and bold lines. B, schematic
-helical representation of amino acid sequence of segment IVS6
( 1S) of chimera AL1 (light gray). High
affinity PAA/BTZ determinants (Tyr1386,
Ala1390, and Ile1393; Refs. 11 and 12), and
heterologous residues of skeletal muscle 1S and rabbit
heart 1C-a sequences in positions 1383 and 1400 are
highlighted in black. C, sequence alignment of
transmembrane segments IVS6 of chimeras AL1 (carp skeletal muscle
1S, see Ref. 32) and AL16 (cardiac L-type
1C-a; see Ref. 17). High affinity determinants of the
PAA receptor site (open boxes) and sequence differences
between 1C-a and 1S (black
boxes) are highlighted. D, IBa of AL1 and
AL16 during depolarizing test pulses from 80 mV to the indicated test
potentials.
|
|
Our recent finding that inactivation determinants in pore lining S6
segments affect use-dependent Ca2+ channel
block by PAA (24) prompted us to examine possible differences in PAA
and BTZ sensitivity between chimeras AL1 and AL16 (Fig. 1D).
Drug sensitivity was characterized as resting state and
use-dependent IBa inhibition (see also Refs. 12
and 25).
Despite the presence of L-type sequence in both constructs
(Fig. 1A), (+)-cis-diltiazem (100 µM) induced
much less IBa inhibition of AL1 compared with the
pronounced block of AL16 (Fig. 2,
A and B). Fig. 2 compares IBa block
of AL1 and AL16 by (
)-gallopamil and (+)-cis-diltiazem. In line with
previous observations, (
)-gallopamil (100 µM) induced
pronounced use-dependent Ca2+ channel block of
AL1 and AL16 (12, 16). Resting state-dependent Ca2+ channel block was more prominent for the
benzothiazepine than for the PAA (Fig. 2B; see Ref. 18 for
similar observations on cardiac myocytes).

View larger version (37K):
[in this window]
[in a new window]
|
Fig. 2.
Sensitivity of AL1 and AL16 for
(+)-cis-diltiazem and ( )-gallopamil. A,
use-dependent block of IBa in AL1 (upper
panel) and AL16 (lower panel) by (+)-cis-diltiazem and
( )-gallopamil was estimated during trains of 15 test pulses (100 ms)
applied from 80 mV to 20 mV at a frequency of 0.1 Hz. Identical pulse
protocols were applied in the absence of drug (control). B,
use-dependent IBa inhibition (in percentages,
means ± S.E., n = 4-9) during 15 pulses (see
A) by 100 µM (+)-cis-diltiazem (shaded
columns) or 100 µM ( )-gallopamil (hatched
columns) compared with current decay in the absence of drug
(white columns) at a holding potential of 80 mV. Resting
state-dependent block (IBa inhibition during
the first pulse in drug; black columns) was more pronounced
for AL16. C and D, IBa recovery from
inactivation of AL1 (C) and AL16 (D) were
measured by a two-pulse protocol in the absence (open
symbols) and presence (filled symbols) of 100 µM (+)-cis-diltiazem. The currents were inactivated by
3-s prepulses to 20 mV. IBa recovery at 80 mV was
measured by applying a sequence of test pulses at various times after
the prepulse (see "Experimental Procedures"). After a two-pulse
experiment the membrane was hyperpolarized to 100 mV for 4 min to
permit recovery from block and inactivation. Continuous
lines are biexponential fits to the mean time courses, with AL1
recovery time constants rec, fast ( ) = 1.9 ± 0.3 (n = 4) and rec, slow ( )=
31 ± 6 (n = 4) versus
rec, fast ( ) =1.1 ± 0.2 (n = 4) and rec, slow ( ) = 35 ± 3 (n = 4) (C) and AL16 recovery time constants
rec, fast ( ) = 2.1 ± 0.3 (n = 4) and rec, slow ( ) = 25 ± 4 (n = 4) versus rec, fast
( ) = 1.2 ± 0.2 (n = 4) and
rec, slow ( ) = 63 ± 10 (n = 4)
(D).
|
|
Thus, the results shown in Fig. 2A suggest that crucial
determinants of the BTZ receptor are missing in the
1S
sequence of AL1. Differences in (+)-cis-diltiazem sensitivity were
further analyzed by comparing the time course of IBa
recovery at rest. Diltiazem slowed IBa recovery mainly by
delaying the slow recovery component (
rec, slow) (Fig.
2, C and D). In line with the more pronounced
use-dependent block, IBa of AL16 recovered at a
significantly slower rate from block by the benzothiazepine than
IBa of AL1 (
rec, slow (AL1) = 35 ± 3 s versus
rec, slow (AL16) = 63 ± 10 s, Fig. 2, A, C, and D).
The fast recovery time constant was not significantly changed
(
rec, fast (AL1) = 1.1 ± 0.2 s
versus
rec, fast (AL16) = 1.2 ± 0.2 s).
Transfer of a BTZ Receptor Determinant from
1C-a to
IVS6 of
1S--
To elucidate the structural basis for
the higher BTZ sensitivity of AL16 (Fig. 2), we transferred
Ile1383 from
1C-a to the
1S-derived IVS6 segment of AL1 and analyzed the
sensitivity of the resulting mutant (L1383I) for (+)-cis-diltiazem.
As shown in Fig. 3, mutation L1383I (Fig.
1B) significantly enhanced the BTZ sensitivity of AL1. The
single leucine to isoleucine substitution in segment IVS6 of AL1
induced the same amount of resting state and use-dependent
block by (+)-cis-diltiazem (100 µM) as in the highly
sensitive chimera AL16 (Fig. 3A). The IC50 value
of use-dependent IBa inhibition of AL16
(IC50 = 70 ± 15 µM) did not
significantly differ from the mutant L1381I (IC50 = 83 ± 20 µM, p > 0.05) but was
significantly different from AL1 (IC50 = 880 ± 65 µM, n
3 for each concentration).

View larger version (26K):
[in this window]
[in a new window]
|
Fig. 3.
Transfer of BTZ sensitivity from IVS6 of
1C-a (AL16) to IVS6 of
1S (AL1). A,
use-dependent IBa inhibition by
(+)-cis-diltiazem (100 µM) of AL1, L1383I and AL16.
Channel block was estimated as described in the legend to Fig.
2A. B, IBa recovery from inactivation
of L1383I was estimated as shown in panels C and
D of Fig. 2. , control; , drug. Continuous
lines are biexponential fits to the mean time courses, with L1383I
recovery time constants rec, fast ( ) = 2.6 ± 0.4 s and rec, slow ( ) = 39 ± 7 s
(n = 4) versus rec, fast
( ) = 1.2 ± 0.3 s and rec, slow ( ) = 59 ± 7 s (n = 4).
|
|
Furthermore, recovery of L1383I in the presence of (+)-cis-diltiazem
was slower than in AL1 and comparable with recovery of AL16
(
rec, slow (L13831I) = 59 ± 7 s, Figs.
2C and 3B). Mutation L1383I transferred not only
the sensitivity for (+)-cis-diltiazem but additionally slowed
inactivation kinetics compared with AL1 (see inset in Fig.
3A, 27 ± 3% during 300-ms test pulse to 20 mV,
n = 6, significantly slower than AL1). The later
finding was also evident from a reduced accumulation of
Ca2+ channels in inactivation during control pulse trains
(white columns in Fig. 3A).
Taken together, the rearrangement of a single methyl group in the outer
part of segment IVS6 (mutation L1383I) increased sensitivity for
(+)-cis-diltiazem by delaying unblock of resting Ca2+
channels. A crucial role of Ile1383 for block of resting
Ca2+ channels by (+)-cis-diltiazem is further supported by
an enhanced resting state-dependent block of L1383I
compared with AL1 (Fig. 3A).
Role of Ile1487 and Val1504 in
(+)-cis-Diltiazem Block of a Cardiac L-type
1 Subunit--
Results in Fig. 3 illustrate that an
amino acid at the outer channel mouth in segment IVS6 may have a
specific effect on (+)-cis-diltiazem binding and dissociation to
resting (closed) channels. Because this finding was obtained using
chimeric channels, which might produce peculiar effects caused by an
altered sequence environment, we subsequently studied the role of both
divergent amino acids in segment IVS6 (Ile1383 and
Val1400) using the cardiac
1C-a subunit
derived construct Lc (see "Experimental Procedures"). As shown in
Fig. 4, substitution of the "cardiac" isoleucine in position 1487 by either the corresponding leucine of the
carp skeletal muscle
1S or an alanine (I1487L or I1487A) significantly reduced use-dependent channel block by
accelerating unblock at rest (Fig. 4, compare unblock of I1487A with
unblock of Lc).

View larger version (36K):
[in this window]
[in a new window]
|
Fig. 4.
Use-dependent block and recovery
of Lc and mutant Lc subunits I1487L, I1487A, and V1504A.
A, comparison of the inactivation time course of Lc and
mutants I1487L, I1487A, and V1504A illustrated during 1-s depolarizing
pulses from 80mV to the indicated test potentials. Mutation V1504A
substantially reduced channel inactivation. Compare with inactivation
kinetics of Lc. B, comparison of use-dependent
IBa block of Lc and derived mutants. IBa
inhibition was measured as cumulative current inhibition (in
percentages) during 15 depolarizing pulses (100 ms) after 3-min
incubation of the Xenopus oocytes in drug (shaded
columns) (see Fig. 2B). Test pulses were applied at a
frequency of 1 Hz. Resting state-dependent block shown by
the black columns. Peak IBa decay under control
conditions is shown by the white columns. Bars
represent the mean ± S.E. (n = 4-8). Statistical
significance compared with IBa block of Lc is indicated by
asterisks. C, IBa recovery from
inactivation in the absence or presence of 100 µM
(+)-cis-diltiazem. Continuous lines are single exponential
fits to the mean recovery time courses of Lc ( , control; , in
drug), I1487A ( , in drug), and V1504A ( , in drug) with recovery
time constants: Lc (control) rec = 0.18 ± 0.04 s (n = 4) versus Lc (drug)
rec = 2.94 ± 0.32 s (n = 4),
I1487L (control, not shown) rec = 0.31 ± 0.12 s (n = 4) versus I1487A (drug)
rec = 1.0 ± 0.06 s (n = 4),
I1487A (control, not shown) rec = 0.27 ± 0.12 s (n = 5) versus I1487A (drug)
rec = 0.97 ± 0.13 s (n = 6),
I1504A (control, not shown) rec = 9.93 ± 1.68 s (n = 3) versus I1504A (drug)
rec = 3.5 ± 0.55 s (n = 3).
D, mean time constants of IBa recovery from
inactivation in control (white columns) compared with
recovery time constants in the presence of 100 µM
(+)-cis-diltiazem (black columns) of constructs Lc, I1487L,
I1487A, and V1504A. Because of its slower inactivation (see panel
A), recovery of mutant V1504A was estimated after 30 s
prepulses. Statistical significance compared with IBa block
of Lc is indicated by asterisks.
|
|
Substituting alanine for valine in position 1504 at the inner channel
mouth (V1504A) dramatically slowed Ca2+ channel
inactivation (Fig. 4A) and simultaneously diminished use-dependent IBa inhibition by
(+)-cis-diltiazem (Fig. 4B). A subsequent analysis of the
IBa recovery kinetics from block clearly demonstrated that
this substitution at the inner channel mouth (V1504A) did not
significantly affect unblock of resting channels (Fig. 4C,
compare unblock of V1504A and Lc).
Kinetics of Open Ca2+ Channel Block by
(+)-cis-Diltiazem--
To investigate the kinetics of
(+)-cis-diltiazem interaction with Ca2+ channels in the
open state, we analyzed the rate and extent of IBa decay at
different drug concentrations during depolarizing test pulses to 20 mV
(26). As for the L-type construct Lc, (+)-cis-diltiazem blockade of mutants I1487A and V1504A occurred with bimolecular association kinetics. The drug association rate
(kon) for all three constructs was proportional
to the drug concentration, and the off rate
(koff) was independent of the applied drug
concentration (Fig. 5). We observed no
differences between the drug association kinetics of Lc and mutant
I1487A. However, V1504A displayed significantly slowed association
kinetics compared with Lc with kon(Lc) = (5.85 ± 0.24) × 103 M
1
s
1 versus kon(V1504A) = (4.93 ± 0.24) × 103 M
1
s
1 (p < 0.005). No significant changes
in koff were observed for the Lc mutants.

View larger version (27K):
[in this window]
[in a new window]
|
Fig. 5.
IBa inhibition of Lc and derived
mutants I1487L, I1487A, and V1504A at 20 mV. The rate constants
(kon and koff) of an open
channel block by (+)-cis-diltiazem were estimated from the current
relaxation time course ( , see single exponential fit on currents in
the inset: 1, control; 2, in 30 µM (+)-cis-diltiazem; 3, in 100 µM (+)-cis-diltiazem; and 4, in 300 µM (+)-cis-diltiazem). The extent of block
(f = Isteady-state/Ictrl) was
estimated during the first test pulse after 3 min incubation of the
oocyte in drug. koff was estimated as f/ .
Data points are the mean values with standard error of 5-7
experiments. Linear regression yielded kon (Lc)=
(5.85 ± 0.24) × 103 M 1
s 1, kon (I1487A)= (6.1 ± 0.14) × 103 M 1 s 1
and kon (V1504A) = (4.93 ± 0.24) × 103 M 1 s 1
(R > 0.98). Mean koff did not
depend on drug concentration and ranged between 0.16 and 0.27 s 1.
|
|
 |
DISCUSSION |
The characteristics of stereospecific and
use-dependent L-type Ca2+ channel
block by diltiazem can be transferred to the lowly sensitive
1A subunit of class A Ca2+ channels by three
L-type specific amino acids that have previously been
identified as high affinity determinants of the PAA receptor site (11,
12). These experiments suggested that the BTZ and PAA receptors have
overlapping but not identical determinants in the Ca2+
channel segment IVS6. Cai et al. (13) reported an
approximately 10-fold increase in the half-maximal inhibitory
concentration of Ca2+ currents by diltiazem if the PAA
receptor determinants in segments IVS6 of an L-type channel
(
1C-a) were replaced by the corresponding
1A amino acids. Here we confirm a key role of segment
IVS6 for Ca2+ channel interaction with (+)-cis-diltiazem
and report additional BTZ-specific receptor determinants in this pore
lining segment (Fig. 2).
Structural Determinants of Resting State-dependent
Action of (+)-cis-Diltiazem--
Our data show that an amino acid
residue (Ile1487), which is located close to the
extracellular mouth of the L-type channel pore, as well as
an inactivation determinant (Val1504;
1C-a
numbering) at the inner channel mouth participate in the regulation of
Ca2+ channel block by BTZs. As shown in Fig. 4, an alanine
or leucine substitution of Ile1487 in Lc (compare also AL1
and L1383I, Fig. 3) substantially accelerated channel unblock at rest
(Figs. 3 and 4) without altering the association or dissociation rate
of (+)-cis-diltiazem for open channels (Fig. 5). The reduction in
use-dependent block in the Lc mutants can therefore be
explained by an enhanced recovery of channels between the individual
pulses of a train rather than by a reduced drug affinity of the
channels in the open state.
Enhanced recovery could be caused by a reduced bulkiness of a single
amino acid resulting in facilitated drug dissociation or alternatively
by a direct interaction of the drug molecule with the amino acid side
chain in the resting state. If the bulkiness of a single isoleucine in
position 1487 would restrict dissociation of (+)-cis-diltiazem from its
receptor, removal of the side chain by the substituting isoleucine with
the substantially smaller alanine is expected to facilitate channel
unblock to a greater extent than exchanging isoleucine for the equally
bulky and hydrophobic leucine. However, both Leu and Ala reduced
use-dependent Ca2+ channel block to a
comparable extent (Fig. 4). Furthermore, channel unblock kinetics of
I1487L and I1487A at rest were indistinguishable and occurred at a
significantly faster rate than unblock of Lc (Fig. 4, C and
D). It is therefore unlikely that the enhanced unblock at
rest is determined by the bulkiness of a single amino acid in position
1487. Instead, a single methyl group appears to form part of the
BTZ-binding pocket of resting closed channels.
It is hard to distinguish whether this methyl group exclusively
contributes to binding of diltiazem to closed resting channels or
additionally forms part of a complex guarding structure. In the
L-type environment of Lc, amino acid substitutions I1487L and I1487A did not significantly decrease resting
state-dependent block (Fig. 4B). Therefore, we
favor a "guarding hypothesis" where I1487 simultaneously forms part
of a guarding structure. It is tempting to speculate that this guarding
structure determines not only channel unblock (Figs. 2, 3, and
4C) but also access of (+)-cis-diltiazem to its binding
determinants (Fig. 6).

View larger version (40K):
[in this window]
[in a new window]
|
Fig. 6.
A guarded and modulated BTZ receptor.
Putative pore orientated amino acids in the central part of
transmembrane segment IVS6 contribute high affinity determinants of the
BTZ and PAA receptor sites (Tyr1490, Ala1494,
and Ile1497; 1C-a numbering; see Refs.
11-13). Amino acid substitutions at position 1487 affect channel
unblock at rest (Figs. 3B and 4, C and
D) and consequently use-dependent block during a
train (Figs. 3A and 4B). Our data suggest that
isoleucine in position 1487 of 1C-a (close to the
extracellular channel mouth) forms part of the BTZ receptor site and
additionally determines dissociation and access of (+)-cis-diltiazem to
its receptor site in the closed resting state. Other inactivation
determinants close to the inner channel mouth in segment IIIS6
(Phe1191 and Val1192; 1C-a
nomenclature) also affect use-dependent Ca2+
channel block by BTZ (15) and PAA (24). Val1504 at the
inner mouth of segment IVS6 is a strong L-type channel
inactivation determinant (Fig. 4A) simultaneously affecting channel
block by (+)-cis-diltiazem (Figs. 4B and 5). The role of
Val1504 in channel block can be explained by an allosteric
modulation of the drug binding reaction. Additional trapping of the
blocker in an inactivated channel state or a direct contribution of
binding energy by Val1504 cannot be excluded.
|
|
Our data suggest that the BTZ receptor is located closer to the
extracellular mouth of the channel pore than the PAA receptor (11, 12),
which is in line with previous findings demonstrating an extracellular
access of quaternary BTZ SQ32,428 (27) compared with the intracellular
access of PAA (25, 28, 29).
Modulation of the BTZ Receptor by a Channel Inactivation
Determinant--
A cluster of homologous inactivation determinants
located close to the inner mouth of the channel pore affecting
Ca2+ channel block by PAA was recently identified in
segment IIIS6 (24). Kraus et al. (14) reported that alanine
substitutions of the same residues in segment IIIS6 of a BTZ-sensitive
class A/L-type channel chimera decreased both current
inactivation and use-dependent block by (+)-cis-diltiazem.
Here we report an analogous role of a residue at the inner mouth of the
pore in segment IVS6 in a recombinant L-type channel. As
shown in Fig. 4A, substituting a single valine in segment
IVS6 by alanine (V1504A) dramatically slowed Ca2+ channel
inactivation kinetics and reduced use-dependent block (Fig.
4, A and B). It is interesting to note that the
inactivation determinant valine in position 1504 of Lc
(Val1504) is conserved in all known Ca2+
channel classes with the exception of the
1S subunit
cloned from carp skeletal muscle (Fig. 1). These findings provide
further evidence for a key role of S6 segments in inactivation gating of Ca2+ channels and confirm a crucial role of homologous
residues that are located at the inner channel mouth (see Ref. 30 for review).
The analysis of (+)-cis-diltiazem interaction with the
inactivation-deficient mutant V1504A at a depolarized potential
revealed a reduced drug association rate (kon)
compared with mutants I1487A and Lc (Figs. 4 and 5). The reduced
use-dependent block of V1504A (Fig. 4B) is
therefore caused by a reduced drug binding reaction at 20 mV and not by
an accelerated channel unblock at rest as observed for I1487L and
I1487A (Figs. 4D and 5). Once the drug gained access to its
receptor determinants in the open state, inactivating channels are in a
more favorable conformation for interaction with (+)-cis-diltiazem than
the "noninactivating" mutant V1504A (Figs. 4A and
5).
A guarded and modulated receptor hypothesis illustrating the impact of
Ile1478 at the outer channel mouth and Val1504
at the inner channel mouth in use-dependent
Ca2+ channel block by (+)-cis-diltiazem is schematically
illustrated in Fig. 6. Ile1487 and Val1504 are
separated by five turns of the
-helical IVS6 segments (
27 Å).
Compared with the short distance of the putative pharmacophores of
diltiazem (between 6 and 9 Å; see Ref. 31) the extended structure of
the BTZ receptor suggests that some of the inactivation determinants at
the inner channel mouth of IIIS6 (15) and IVS6 (Fig. 4) affect Ca2+ channel block not by contributing binding energy but
via allosteric modulation of the drug binding process.
However, neither an additional drug trapping of (+)-cis-diltiazem in an
inactivated channel state (24) nor a direct contribution of binding
energy by Val1504 can be excluded (Fig. 6).
In summary, we describe for the first time determinants of the BTZ
receptor site that selectively affect BTZ interaction with Ca2+ channels in resting and open or inactivated states. We
have demonstrated that a mutation at the inner channel mouth reducing
channel inactivation (V1504A) simultaneously reduces the affinity of
the BTZ binding site at depolarized potentials. Mutations at the
external mouth of the pore (I1487L and I1487A) modulate drug
dissociation without significantly affecting the drug affinity of open
channels. Further mutational analysis of drug receptor sites will
require a differentiation between drug access pathways, drug-binding
sites, and conformational changes during membrane depolarization
(e.g. inactivation).