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Department of Anesthesiology, Friedrich-Alexander-University Erlangen-Nuremberg, Erlangen, Germany (C.N.); Department of Biological Sciences, State University of New York at Albany, Albany, New York (S.-Y.W.); and Department of Anesthesia Research Laboratories, Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts (G.K.W.)
Received October 8, 2002; accepted March 3, 2003
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Abstract |
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 Top Abstract Materials and MethodsResultsDiscussionReferences |
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-subunit comprises four homologous domains (D1 to D4), each containing
six -helical transmembrane segments (S1 to S6) and an S5-S6 linker that
forms a P-loop as a hairpin from the extracellular side. These P-loops line
the outer part of the channel's pore and form the selectivity filter, whereas
the S6 segments of each domain are believed to line the inner part of the
channel's pore (Catterall,
2000
). The highly positively charged S4 segments in each domain
are postulated to serve as a voltage sensor
(Stuhmer et al., 1989;
Kontis et al., 1997). Their
outward movements upon depolarization
(Yang and Horn, 1995;
Yang et al., 1996) are
believed to induce conformational changes in the pore that result in channel
activation. During the activation process, S6 segments are likely to exhibit
rotational/lateral movements, as suggested for K+ channels
(Perozo et al., 1999;
Jiang et al., 2002). Segments
D3-S4 and D4-S4 were proposed to be important for coupling channel activation
to fast inactivation (Cha et al.,
1999; Sheets et al.,
1999). The structural determinants of Na+ channel fast
inactivation are hydrophobic residues (Ile-Phe-Met) in the intracellular D3-D4
loop that serve as an inactivation gate
(Vassilev et al., 1988;
West et al., 1992;
Eaholtz et al., 1994) and
several residues at the intracellular end of D4-S6 (McPhee et al.,
1994,
1995) and within intracellular
S4-S5 loops of D3 (Smith and Goldin,
1997) and D4 (McPhee et al.,
1998) that serve as an inactivation gate receptor.
LAs block the propagation of action potential by binding in the
ion-conducting pore of voltage-gated Na+ channels. LAs more avidly
bind to open and inactivated channel states rather than to resting channel
states, suggesting a binding site that converts from a low- to a high-affinity
conformation during state transitions of the channel
(Hille, 1977;
Hondeghem and Katzung, 1977).
Mapping of the LA binding site by means of alanine-scanning mutagenesis of the
rat brain Nav1.2 channel and electrophysiological experiments in
the oocyte expression system revealed specific amino acid residues in segments
D4-S6, D3-S6, and D1-S6 that are involved in binding of the LA etidocaine. The
two hydrophobic aromatic residues phenylalanine (F1764) and tyrosine (Y1771)
in D4-S6 (Ragsdale et al.,
1994) and the amino acid residues L1465, N1466, and I1469 in D3-S6
(Yarov-Yarovoy et al., 2001)
have been proposed to face the channel pore and to form the high-affinity
binding site for this LA. Moreover, L1465, N1466, and I1469 in D3-S6 were
suggested to be involved in voltage-dependent activation and coupling to
closed-state inactivation. Recently, residue I409 in D1-S6 was proposed to
also contribute minimally to the LA binding site
(Yarov-Yarovoy et al., 2002),
whereas none of the residues in D2-S6 was found to do so
(Wang et al., 2001;
Yarov-Yarovoy et al.,
2002).
Lysine point mutations of the rat skeletal muscle Nav1.4 channel
expressed in human embryonic kidney (HEK) 293t cells confirmed the
contribution of phenylalanine F1579 in D4-S6 (homologous to F1764 in
Nav1.2) to the binding of various LAs
(Wright et al., 1998) and
found S1276 and L1280 in D3-S6 (homologous to S1461 and L1465 in
Nav1.2) to be involved in binding of the enantiomers of the LA
bupivacaine (Wang et al.,
2000). In addition, N434 and L437 in D1-S6 were proposed to be
critical for LA binding as well (Wang et
al., 1998). A subsequent study creating mutations at N434 that
vary the hydrophobicity, aromaticity, polarity, and charge at this site
revealed an intriguing correlation between the physicochemical properties of
the substituted residues and the changes in potency of bupivacaine enantiomers
to block inactivated channels, suggesting a direct interaction of N434 with
the positively charged moiety of LAs. Supporting this hypothesis was the
finding that mutation N434R exhibited significant stereoselectivity for the
block of inactivated channels that resulted from a selective decrease in block
by S(–)-bupivacaine (Nau et
al., 1999).
In the present work, we carried out a systematic analysis of the role of
residue L1280 in D3-S6 of rat skeletal muscle Nav1.4 channel in
gating and block by the enantiomers of bupivacaine using a similar approach.
Residue L1280 was chosen because both alanine and lysine mutations at this
position had the most robust impact on binding of pore-blocking drugs to
inactivated channels in previous studies
(Wang et al., 2000;
Yarov-Yarovoy et al., 2001).
We substituted the native leucine by a series of amino acid residues that vary
the physicochemical properties at this site. Wild-type and mutant channel
-subunits were expressed transiently in HEK293t cells and were
investigated under whole-cell voltage clamp. As LA probes, we chose the
enantiomers of bupivacaine because stereoisomers can been useful tools in
receptor mapping. Figure 1
shows computer-generated three-dimensional molecular structures of
R(+)-and S(–)-bupivacaine. The results of this study
support the idea that in inactivated channels, residues L1280 in D3-S6 and
N434 in D1-S6 both interact directly with LAs and thereby face each other in
the ion-conducting pore.
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Materials and Methods |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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). HEK 293t cells were transfected with
Nav1.4-pcDNA1 (5 µg) and reporter plasmid CD8-pih3m (1 µg) by
the calcium phosphate precipitation method. After incubation for 12 to 15 h,
cells were replated in 35-mm culture dishes. Transfected cells were used for
experiments within 3 days. Transfection-positive cells were identified by
immunobeads (CD-8 Dynabeads; Dynal Biotech, Oslo, Norway). Chemicals and Solutions. R(+)- or S(–)-bupivacaine were gifts from Dr. Lars-Inge Olsson (AstraZeneca, Södertälje, Sweden). The drugs were dissolved in dimethyl sulfoxide to give stock solutions of 100 mM. The highest dimethyl sulfoxide concentration obtained was 1% and had no effect on Na+ currents. Experiments were performed with an external solution containing 65 mM NaCl, 85 mM choline Cl, 2 mM CaCl2, and 10 mM HEPES (adjusted to pH 7.4 with tetramethylammonium hydroxide) and a pipette solution containing 100 mM NaF, 30 mM NaCl, 10 mM EGTA, and 10 mM HEPES (adjusted to pH 7.2 with CsOH). The reversed Na+ gradient was used to minimize the series resistance artifact, which is less serious with outward currents. After a gigaohm seal and a whole-cell voltage clamp were established, the cells were dialyzed for at least 15 min before data acquisition. Control and test solutions were applied with a multiple-barrel perfusion system.
Electrophysiological Technique and Data Acquisition. Na+
currents expressed transiently in HEK293t cells were recorded at room
temperature with the whole-cell configuration of the patch-clamp method. Patch
pipettes were pulled from borosilicate glass tubes (TW150F-3; World Precision
Instruments, Berlin, Germany) and heat-polished at the tip to give a
resistance of 1.0 to 1.5 M
. Currents were recorded with an Axopatch
200B patch-clamp amplifier (Axon Instruments, Union City, CA), filtered at 5
kHz, and sampled at 20 kHz. Experiments were conducted under capacitance and
series-resistance compensation. Leakage currents were subtracted by the
P/–4 method. pCLAMP 8.0.1 software (Axon Instruments) was used for
acquisition and analysis of currents. Microcal Origin 6.1 software (OriginLab
Corp, Northampton, MA) was used to perform least-squares fitting and to create
figures. Data are presented as mean ± S.E.M. or fitted value ±
S.E. of the fit. An unpaired Student's t test (SigmaStat; SPSS
Science, Chicago, IL) was used to evaluate the significance of changes in mean
values. P values <0.05 were considered statistically
significant.
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Results |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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Wild-type and mutant channel -subunits were expressed transiently in
HEK293t cells and were investigated under whole-cell voltage clamp. Current
density for wild-type was 253 ± 51 pA/pF. Mutants L1280A, L1280C,
L1280D, L1280E, L1280K, L1280N, L1280Q, L1280R, L1280T, and L1280W expressed
sufficient Na+ currents for further analysis, whereas mutants
L1280F and L1280Y expressed little or no Na+ currents. Current
densities for sufficiently expressing mutants all were significantly smaller
compared with wild-type except for mutants L1280C and L1280N
(Table 1). Current densities
did not correlate with the volume or hydropathy index of substituted amino
acids.
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None of the mutants at L1280 tested showed any significant sustained current at the end of a 5-ms depolarization; they all inactivated completely from the open state. The midpoint voltages of steady-state inactivation were significantly shifted leftward in all mutants by approximately –7to –12 mV compared with wild-type (Fig. 2B; Table 1).
Effect of Point Mutations at L1280 on State-Dependent Block by
Bupivacaine Enantiomers. For all mutant channels, steady-state block by
bupivacaine enantiomers was initially assessed with a three-step pulse
protocol as described previously (Nau et
al., 1999), allowing for direct estimation of block of resting and
inactivated channels by LA drugs that unbind slowly from inactivated channels.
In brief, Na+ currents were evoked by 5-ms test pulses to +50 mV
after 10-s conditioning prepulses between –180 and –50 mV (to
allow binding to reach steady-state) and 100-ms intervals at the holding
potential of –140 mV (to allow drug-free channels to recover from fast
inactivation). Figure 3 shows
normalized peak currents of wild-type and selected mutants in control and in
the presence of 10 µM R(+)- or S(–)-bupivacaine as
a function of the conditioning prepulse potential. As described previously for
wild-type channels, slow inactivation was detectable in control after
prepulses 
–100 mV. Block by 10 µM R(+)- or
S(–)-bupivacaine reached plateaus after prepulses
–120 and –70 mV, corresponding to the block of resting and
inactivated channels, respectively. No stereoselectivity was detectable in the
resting state, and little stereoselectivity was detectable in the inactivated
state.
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Mutant currents in control solution exhibited different degrees of slow
inactivation after prepulses –110 mV. This phenomenon is similar to
that observed for mutations at N434, but it likewise did not correlate with
the potency of bupivacaine to block inactivated channels.
Block of resting mutant channels by R(+)- or S(–)-bupivacaine revealed little differences compared with wild-type channels. Block of inactivated channels by R(+)- or S(–)-bupivacaine was slightly increased in mutant L1280W and was dramatically decreased in mutant L1280K. Surprisingly, mutants L1280E, L1280N, L1280Q, and L1280R exhibited significant stereoselectivity for block of inactivated channels. More surprisingly, stereoselectivity resulted from a selective decrease in the block of inactivated channels by R(+)-bupivacaine. This is in contrast to mutation N434R of D1-S6, in which stereoselectivity for block of inactivated channels resulted from a selective decrease in block by S(–)-bupivacaine.
To determine more accurately the potencies of bupivacaine enantiomers in blocking resting and inactivated channels, IC50 values for block of wild-type and mutant channels by R(+)- and S(–)-bupivacaine were derived from concentration-inhibition experiments. A prepulse potential of –140 mV was deemed sufficient to estimate bupivacaine potency for resting channels. Potencies for inactivated channels were all estimated with a prepulse potential of –70 mV. At this potential, block of L1280E, L1280N, L1280Q, and L1280R did not yet reach a plateau, so we might have slightly underestimated the block of inactivated channels in these mutants. The effects of 10 µM R(+)- and S(–)-bupivacaine on wild-type and selected mutant Na+ currents elicited after prepulses to –140 and –70 mV are shown in Fig. 4A, and the IC50 values derived from concentration-inhibition experiments are summarized in Fig. 4, B and C. The channels are arranged from top to bottom traces (Fig. 4A) and from left to right (Fig. 4, B and C) according to the hydropathy index of the substituted amino acid residue, beginning with the most hydrophobic. All IC50 values are listed in Table 2, along with calculated ratios for stereoselective potencies.
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In resting channels, the blocking potency of
S(–)-bupivacaine was slightly increased in L1280A, L1280T, and
L1280D. Blocking potency of R(+)-bupivacaine was slightly decreased
in L1280E, L1280K, and L1280R. In inactivated channels, the blocking potency
of bupivacaine enantiomers was increased only in mutant L1280W. The blocking
potency of R(+)-bupivacaine was selectively decreased in mutants
L1280A, L1280N, L1280Q, L1280D, and L1280E. The blocking potency of both
enantiomers was decreased in mutants L1280K and L1280R. Significant
stereoselectivity was revealed in mutants L1280W, L1280A, L1280N, L1280Q,
L1280D, L1280E, and L1280R and was greatest in mutants L1280R
[R(+)/S(–) = 7], L1280E
[R(+)/S(–) = 5], L1280Q
[R(+)/S(–) = 4], and L1280N
[R(+)/S(–) = 2]. Except for mutation L1280W,
stereoselectivity resulted from a selective decrease in block of inactivated
channels by R(+)-bupivacaine. As mentioned previously, this is in
contrast to the findings in mutation N434R of D1-S6, in which
stereoselectivity for block of inactivated channels resulted from a selective
decrease in block by S(–)-bupivacaine
(Nau et al., 1999). In mutant
N434R, the charge, size, and orientation of atoms and bonds in the guanidinium
group of arginine were held responsible for bupivacaine stereoselectivity.
Effects of substituting native asparagine in position N434 by glutamate and
glutamine were not investigated in that study.
Effects of Mutations N434E and N434Q on Activation, Steady-State Inactivation, and State-Dependent Block by Bupivacaine Enantiomers. To test whether glutamate and glutamine are likewise able to cause bupivacaine stereoselectivity while residing in position N434, we created mutations N434E and N434Q and studied their gating properties and state-dependent interaction with bupivacaine enantiomers.
Activation and inactivation properties of mutations N434E and N434Q were characterized with standard pulse protocols as described in Fig. 2. Original current traces of N434E and N434Q recorded to estimate activation properties are shown in Fig. 5, A and B, respectively, and the corresponding activation curves are shown in Fig. 5C. Mutation N434E exhibited rectification at positive voltages and significant sustained currents at the end of a 5-ms depolarization (Fig. 5A). The midpoint voltage of activation was shifted leftward by –6 mV compared with wild-type channels. Activation properties of N434Q were unchanged compared with wild-type channels. The midpoint voltage of steady-state inactivation was unchanged in mutant N434E and was shifted rightward by 10 mV in mutant N434Q compared with wild-type channels (Fig. 5D; Table 1).
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The effects of 10 µM R(+)- and S(–)-bupivacaine
on mutants N434E and N434Q elicited after prepulses to –140 and
–70 mV are shown in Fig. 6, A and
B. The normalized peak currents of mutant N434E and N434Q in
control and in the presence of 10 µM R(+)-bupivacaine and
S(–)-bupivacaine as a function of different conditioning
prepulse potentials are shown in Fig. 6, C
and D. Significant slow inactivation was detectable in control
channels after prepulses –90 and –80 mV, respectively,
that was more pronounced than in L1280 mutant channels. Block of resting
channels by R(+)- or S(–)-bupivacaine was decreased in
mutant N434E and was unchanged in mutant N434Q. In mutant N434E, block of
inactivated channels by S(–)-bupivacaine was selectively
decreased, resulting in a significant stereoselectivity. In mutant N434Q,
block of inactivated channels was decreased for both bupivacaine enantiomers
compared with wild-type channels. In both mutants, block after prepulses of
–50 mV was less than after prepulses of –70 mV. This decrease in
block could be caused by a lower affinity of slow inactivated channels than
fast inactivated channels for bupivacaine. Alternatively, a modest amount of
channel activation induced by prepulses >–70 mV could result in
knockout of some drug molecules by external Na+ ions. The
IC50 values obtained from concentration-inhibition experiments in
these mutations are listed, along with mutations in L1280, in
Table 2.
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Discussion |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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Role of Residue L1280 in Voltage-Dependent Activation and Steady-State
Inactivation. Alanine mutations in the inner two thirds of D3-S6 were
shown previously to cause periodic positive and negative shifts in the voltage
dependence of activation (Yarov-Yarovoy et
al., 2001). Mutation L1465A in Nav1.2 channels
(homologous to L1280 in Nav1.4 channels), causing a positive shift,
was reasoned to belong to a group of mostly hydrophobic residues on one side
of the -helical segment D3-S6 that faces the lumen of the pore in the
activated and inactivated state and interacts with surrounding transmembrane
segments in D3 in the resting state
(Yarov-Yarovoy et al.,
2001).
We show here that all mutations at L1280 cause positive shifts in the
voltage-dependent activation and negative shifts in steady-state inactivation.
However, our data do not reveal a clear relationship between shifts in
activation or steady-state inactivation and the physicochemical properties of
the substituted residues. These findings may indicate that the native
aliphatic nonpolar residue leucine at position L1280 is involved in a highly
specific intramolecular interaction that is required for normal activation and
inactivation gating. Furthermore, our data do not reveal a significant
correlation between positive shifts of activation and negative shifts of
steady-state inactivation (Fig. 2, C and
D). Thus, residue L1280 is differentially involved in the process
of activation and inactivation. The incomplete inactivation from the open
state observed in mutation N434E is consistent with the proposed importance of
residues at the intracellular end of segment D1-S6, among others, for closure
of the fast inactivation gate
(Yarov-Yarovoy et al., 2002).
The peculiar rectification at positive voltages in mutation N434E
(Fig. 5, A and C) might be
caused by an interference of the glutamate residue with intracellular
Na+ ions and supports the idea that residue N434 is located close
to the intracellular entry of the pore.
Role of Residue L1280 in State-Dependent Block by Bupivacaine
Enantiomers. In this study, block of resting and inactivated channels by
bupivacaine enantiomers could be clearly separated and directly measured in
both wild-type and mutant channels. As demonstrated previously, LA affinities
of resting and inactivated channels measured at strongly negative
(–140 mV) and less negative potentials (–70 mV),
respectively, are not affected by shifts in the voltage dependence of
steady-state inactivation (Wright et al.,
1999). Block of inactivated channels was measured after 10-s
conditioning prepulses to –70 mV, which induced some degree of slow
inactivation in wild-type and mutant channels. Thus, slow inactivated channels
may have contributed to the block designated as block of inactivated channels.
However, similar to mutations at N434 in D1-S6, shifts in fast and slow
inactivation in mutations at L1280 in D3-S6 did not correlate with changes in
block by bupivacaine enantiomers.
As shown previously for mutations at N434 in D1-S6
(Nau et al., 1999) and other
mutations of residues presumably involved in LA binding in D3-S6
(Yarov-Yarovoy et al., 2001)
and D4-S6 (Ragsdale et al.,
1994), mutations at L1280 in D3-S6 affected block of resting and
inactivated channels by bupivacaine enantiomers differently. Most resting
mutant channels showed a higher affinity for bupivacaine than did resting
wild-type channels. For alanine mutations in D3-S6, it was suggested that in
resting channels, additional space is created for the drug molecule to reach
its binding site (Yarov-Yarovoy et al.,
2001). Our findings give additional support to the idea that the
orientation of the side chain of the residues is likely to change in response
to gating movements of S6 segments so that LAs might interact differently with
relevant residues depending on channel state, as suggested for residues in
D4-S6 (Li et al., 1999).
Mutations in N434 revealed an intriguing correlation between the
physicochemical properties of the substituted residues and the changes in
potency of bupivacaine enantiomers to block inactivated channels, consistent
with a direct interaction of N434 with the positively charged amino group of
bupivacaine (Nau et al.,
1999). Bupivacaine block of inactivated channels was enhanced for
mutations carrying an aromatic residue and a negatively charged residue,
presumably through cation-
electron and electrostatic interaction with the
positively charged moiety of bupivacaine, respectively. Bupivacaine block of
inactivated channels was decreased for mutations carrying a positively charged
residue, presumably through electrostatic charge-charge repulsion.
The data for mutations at L1280 show striking similarities to those for mutations at N434. Bupivacaine block of inactivated channels was likewise enhanced in mutation L1280W carrying an aromatic residue. Unfortunately, two other mutations of that type, L1280F and L1280Y, did not express sufficient current for further analysis. Bupivacaine block of inactivated channels was likewise decreased in mutations L1280K and L1280R carrying a positively charged residue. These finding suggest an interaction of residue L1280 in D3-S6 with LAs similar to that of residue N434 in D1-S6.
However, in contrast to mutation N434D, bupivacaine block of inactivated channels was not increased in mutation L1280D but was unchanged for S(–)-bupivacaine and slightly decreased for R(+)-bupivacaine. Mutation L1280E, carrying another negatively charged but larger residue, revealed a similar pattern. Here, block by R(+)-bupivacaine was substantially decreased, resulting in a significant bupivacaine stereoselectivity. A comparable result was obtained for mutation N434E, in which block by S(–)-bupivacaine was selectively decreased, resulting in a moderate bupivacaine stereoselectivity. These data indicate that, besides electrostatic interactions at both positions L12380 [GenBank] in D3-S6 and N434 in D1-S6, the size and the chemical structure of the residue might also play an important role in binding interactions. Altogether, data from mutations at either N434 or L1280 provide compelling evidence that, in the inactivated state, residues N434 and L1280 directly interact with LAs.
It is interesting that mutation L1465A in the rat brain Nav1.2
channel, which corresponds to L1280A in the rat skeletal muscle
Nav1.4 channel, showed significantly reduced affinity of
inactivated channels for the LA etidocaine in electrophysiological experiments
in the oocyte expression system
(Yarov-Yarovoy et al., 2001).
In our experiments, block of resting and inactivated channels by bupivacaine
enantiomers in mutation L1280A was only slightly changed compared with
wild-type channels. Several explanations may account for these differences.
First, homologous residues in different Na+ channel isoforms,
specifically in Nav1.2 and Nav1.4, may play disparate
roles in LA binding. Second, the type of expression system might affect the
actions of LAs on Na+ channels. For example, differences between
expression systems may reflect different post-translational modifications of
channels. Third, leucine at position L1280/L1465 may interact differentially
with drug-specific moieties in LA molecules such as etidocaine and
bupivacaine.
There are two striking differences in stereoselectivity induced by mutations at positions L1280 and N434. First, stereoselectivity was more pronounced in mutations at L1280. Second, stereoselectivity in L1280 was caused by a selective decrease in block by R(+)-bupivacaine, whereas in N434, it was caused by a selective decrease in block by S(–)-bupivacaine.
The amount of stereoselectivity [R(+)/S(–)] for L1280R, L1280E, L1280Q, and L1280N was 7, 5, 4, and 2, respectively. The amount of stereoselectivity [S(–)/R(+)] for N434R, N434E, and N434Q was 3, 2.5, and 1.1, respectively. This suggests that in inactivated channels, the chiral part of a bupivacine molecule may be closer to L1280 in D3-S6 than to N434 in D1-S6.
The minimal structural requirement for a drug-receptor interaction to
express stereoselectivity is involvement of three groups linked to the chiral
center of a drug in interaction with the binding site. Consistent with these
requirements, a bupivacaine molecule interacts with residues in D1-S6, D3-S6,
and D4-S6 of the Na+ channel. The most critical component of the
high-affinity local anesthetic binding site might be segment D4-S6, containing
the aromatic residue phenylalanine. Mutations of this phenylalanine caused the
most dramatic reduction in the block of inactivated channels
(Ragsdale et al., 1994;
Wright et al., 1998).
Furthermore, segment D4-S6 might be the least mobile of all S6 segments
because of the lack of a critical glycine residue responsible for the
flexibility of other S6 segments in the channel opening
(Jiang et al., 2002).
We propose that the guanidinium group of arginine, the carboxylic acid moiety of glutamate, or the acid amide moiety of glutamine introduce a steric hindrance at both positions N434 in D1-S6 and L1280 in D3-S6 so that the inhibitory effect of either R(+)- or S(–)-bupivacaine is disrupted, depending on which segment is carrying the mutation. In other words, only one of the enantiomers is able to align appropriately at the binding site in the presence of a steric hindrance at position L1280 or N434 (Fig. 7).
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This hypothesis implies that the moieties of a bupivacine molecule interacting with D1-S6 and D3-S6 are interchangeable whereas the moiety interacting with D4-S6 is not, and, more importantly, that N434 in D1-S6 and L1280 in D3-S6 face each other while interacting with LAs in the inactivated state, but not necessarily in the resting state. The increase in LA affinity during inactivation might be entirely caused by the alignment of these two residues.
In conclusion, our data provide more evidence that in addition to residues in segment D4-S6, residues N434 in D1-S6 and L1280 in D3-S6 both contribute to the high-affinity binding site for local anesthetics in inactivated Na+ channels.
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Acknowledgements |
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Footnotes |
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ABBREVIATIONS: LA, local anesthetic; HEK, human embryonic kidney.
Address correspondence to: Dr. Carla Nau, Department of Anesthesiology, Friedrich-Alexander-University Erlangen-Nuremberg, Krankenhausstr. 12, 91054 Erlangen, Germany. E-mail: Carla.Nau{at}kfa.imed.uni-erlangen.des
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) were normalized to
the current obtained with a prepulse to –180 mV. The control data for
experiments with R(+)-and S(–)-bupivacaine were
combined. Currents obtained in the presence of 10 µM R(+)-
(
) or S(–)-bupivacaine (
) were normalized to the
current obtained in control with the corresponding prepulse potential. Solid
lines represent fits of the data to a Boltzmann function.
