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Vol. 56, Issue 2, 404-413, August 1999
Department of Anesthesia Research Laboratories, Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts (C.N., G.R.S, G.K.W.), and Department of Biological Sciences, State University of New York at Albany, Albany, New York (S.-Y.W.)
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Summary |
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 Top
 Summary
 Introduction
Materials and Methods
Results
Discussion
References
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Voltage-gated Na+ channels are the primary targets of
local anesthetics (LAs). Amino acid residues in domain 4, transmembrane segment 6 (D4-S6) form part of the LA binding site. LAs inhibit binding
of the neurotoxin batrachotoxin (BTX). Parts of the BTX binding site
are located in D1-S6 and D4-S6. The affinity of BTX-resistant Na+ channels mutated in D1-S6 (µ1-N434K, µ1-N437K)
toward several LAs is significantly decreased. We have studied how
residue µ1-N434 influences LA binding. By using site-directed
mutagenesis, we created mutations at µ1-N434 that vary the
hydrophobicity, aromaticity, polarity, and charge and investigated
their influence on state-dependent binding and stereoselectivity of
bupivacaine. Wild-type and mutant channels were transiently expressed
in human embryonic kidney 293t cells and investigated under
whole-cell voltage-clamp. For resting channels, bupivacaine enantiomers
showed a higher potency in all mutant channels compared with wild-type
channels. These changes were not well correlated with the physical
properties of the substituted residues. Stereoselectivity was small and
almost unchanged. In inactivated channels, the potency of bupivacaine was increased in mutations containing a quadrupole of an aromatic group
(µ1-N434F, µ1-N434W, µ1-N434Y), a polar group (µ1-N434C), or a
negative charge (µ1-N434D) and was decreased in a mutation containing
a positive charge (µ1-N434K). In mutation µ1-N434R, containing the
positively charged arginine, the potency of S(
)-bupivacaine was
selectively decreased, resulting in a stereoselectivity (stereopotency ratio) of 3. Similar results were observed with cocaine but not with RAC 109 enantiomers. We propose that in inactivated channels, residue µ1-N434 interacts directly with the positively charged moiety
of LAs and that D1-S6 and D4-S6 form a domain-interface site for
binding of BTX and LAs in close proximity.
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Introduction |
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Top
Summary
Introduction
Materials and Methods
Results
Discussion
References
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Na+
channels are transmembrane proteins that govern voltage-dependent
modulation of Na+ ion permeability of excitable
membranes. Mammalian Na+ channels consist of one
large 
subunit and one or two smaller 
subunits. The subunit
is formed by four homologous domains (D1-D4), each containing six
transmembrane segments (S1-S6; Catterall, 1995
).
Local anesthetics (LAs) block the propagation of action potentials in
excitable membranes by binding to voltage-gated
Na+ channels. LA binding, as defined by the
potency for inhibiting ionic Na+ current, is
modulated by channel state. Resting channels have a low affinity for
LAs; open and inactivated channels have a higher affinity. The changes
between low- and high-affinity channels can be explained by
voltage-dependent conformational changes of the LA binding site
(modulated receptor hypothesis: Hille, 1977; Hondeghem and Katzung,
1977).
Amino acid residues in D4-S6 have been identified as molecular
determinants of LA binding (Ragsdale et al., 1994). Mutations F1764A
and Y1771A of rat brain IIA Na+ channels had the
strongest effect in decreasing the affinity of open and inactivated
channels toward the LA etidocaine. Because these residues are oriented
on the same face of the helix, it was suggested that they face the
channel pore and that they interact with LA molecules through
hydrophobic or cation-
electron interactions. The selectivity filter
of Na+ channels was shown to participate in
antiarrhythmic drug binding and access to the site as well and
therefore was located adjacent to D4-S6 (Sunami et al., 1997).
The neurotoxin batrachotoxin (BTX) also binds to voltage-gated
Na+ channels. Upon binding, BTX shifts the
activation process toward hyperpolarizing potentials and inhibits fast
and slow inactivation. LAs are allosteric inhibitors of BTX binding.
Interactions with the closed state of BTX-modified
Na+ channels are hampered for LAs, but they bind
readily to the open state (Wang and Wang, 1992) without displacing BTX
from its receptor (Zamponi et al., 1993).
Photoaffinity labeling (Trainer et al., 1996) and subsequent
site-directed mutagenesis studies (Wang and Wang, 1998) have revealed
three residues in D1-S6 that are probably involved in BTX binding: the
conserved µ1-N434 residue and the two adjacent residues µ1-I433 and
µ1-L437 (Wang and Wang, 1998). Mutations µ1-I433K, µ1-N434K,
µ1-N434R, and µ1-L437K rendered the Na+
channels completely insensitive to BTX. A recent study investigating the interactions of BTX with molecular determinants of the LA binding
site showed that parts of the BTX binding site are located in D4-S6 as
well (Linford et al., 1998), sharing overlapping but nonidentical
molecular determinants with the LA binding site in D4-S6. A
domain-interface allosteric model of BTX binding, similar to the
domain-interface model of Ca2+-channel drug
binding (interface of D3-S6 and D4-S6; Hockerman et al., 1997), was
proposed for BTX binding to the Na+ channel, with
its dimethylpyrrolidone carboxylic acid group directed toward D1-S6 and
its steroid moiety toward D4-S6.
A study investigating LA action on BTX-resistant µ1
Na+ channels showed that LA binding was reduced
in mutations µ1-N434K and µ1-L437K (Wang et al., 1998), most
strongly for the charged LA QX314 and minimally for the neutral LA
benzocaine. In mutation µ1-N434D, the binding affinity was increased
for several LAs at a holding potential of 100 mV. The investigators
concluded that residues at the putative BTX binding site in D1-S6 are
critical for LA binding as well and that D1-S6 and D4-S6 align
adjacently along the Na+ permeation pathway.
With the present study, we sought to investigate more rigorously how
residues at the putative BTX binding site in D1-S6 interact with local
anesthetics. We used site-directed mutagenesis to make a series of
amino acid substitutions in position µ1-N434 that vary the
hydrophobicity, aromaticity, polarity, and charge at this site. As
local anesthetic probes, we chose the enantiomers of bupivacaine,
cocaine, and RAC 109, because stereoisomers have been useful tools in
receptor recognition and receptor mapping. However, unmodified
Na+ channels display only weak stereoselectivity
toward bupivacaine enantiomers (Wang and Wang, 1992). On the other
hand, the affinity of BTX-modified channels for S()-bupivacaine
is 30 times higher than that for R(+)-bupivacaine (Wang and Wang,
1992), which suggests that, in BTX-activated channels, the chiral part
of bupivacaine might be oriented toward the BTX binding site or toward
sites that are allosterically changed in the presence of BTX. The
results of this study support the idea that in inactivated channels,
residue µ1-N434 in D1-S6 interacts directly with the positively
charged moiety of LAs.
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Materials and Methods |
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Top
Summary
Introduction
Materials and Methods
Results
Discussion
References
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Site-Directed Mutagenesis and Transient Expression.
The
methods of site-directed mutagenesis have been described (Wang and
Wang, 1997). Mutagenesis of µ1 was performed with µ1-pcDNA1/amp by
means of the Transformer Site-Directed Mutagenesis Kit (Clontech, Inc.,
Palo Alto, CA). Two primers (a mutagenesis primer and a restriction
primer) were synthesized and used to generate the desired mutants. The
restriction primer was 5'-CTGGCGGCCGGTCGACCATGCATCTAG-3', in
which the wild-type XhoI site has been changed to a
SalI site (italic). In vitro synthesis was performed for a
total of 4 h, with one addition of deoxynucleoside triphosphates
and T4-DNA polymerase during the reaction. The potential mutants were
identified by restriction enzyme digestion and confirmed by DNA
sequencing with appropriate primers near the mutated region.
). Cells were grown
to 50% confluence in a Ti25 flask (Falcon 25 cm2/50
ml; Becton Dickinson Labware, Franklin Lakes, NJ) for transfection. After transfection of µ1-pcDNA1 (10 µg) and reporter plasmid
CD8-pih3 m (1 µg) for 15 h, cells were replated in 35-mm culture
dishes. Transfected cells were used for experiments within 4 days.
Transfection-positive cells were identified by immunobeads (CD-8
Dynabeads; Dynal, Lake Success, NY).
Chemicals and Solutions. Bupivacaine and RAC 109 enantiomers were gifts from Chiroscience Ltd. (Cambridge, UK), Dr. Rune Sandberg (ASTRA Pain Control, Södertälje, Sweden), and the late Dr. Bertil Takman (Astra Pharmaceutical Products, Inc., Worcester, MA). Cocaine enantiomers were obtained from Dr. Rao Rapaka (National Institute on Drug Abuse, Bethesda, MD).
The drugs were dissolved in aqueous solution to give 100-mM stock solutions and were stored at20°C. Experiments were performed with
an external solution containing 150 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 than with inward currents (Cota and Armstrong, 1989).
After a gigaohm seal and a whole-cell voltage-clamp were established,
the cells were dialyzed for a minimum of 20 min before data were
acquired. Control solutions as well as test solutions were applied with
a multiple-barrel perfusion system.
Electrophysiological Technique and Data Acquisition.
Na+ currents transiently expressed in HEK 293t
cells were recorded at room temperature (23 ± 2°C) with the
whole-cell configuration of the patch-clamp method (Hamill et al.,
1981). Patch pipettes were pulled from borosilicate glass tubes
(TW150F-3; World Precision Instruments, Sarasota, FL) and heat-polished
at the tip to give a resistance of 0.6-1.0 M
. Currents were
recorded with an Axopatch 200A patch clamp amplifier (Axon Instruments,
Foster City, CA), filtered at 5 kHz, sampled at 20 kHz, and stored on
the hard disk of an IBM-compatible computer. All experiments were
conducted under capacitance cancellation and series-resistance
compensation. Series-resistance errors were about 5 mV on average after
compensation. Leakage currents were subtracted by the P/-4 method.
Liquid junction potentials were <3 mV and were not corrected.
pCLAMP 6.0 software (Axon Instruments) was used for acquisition and
analysis of currents. Microcal Origin software (Microcal Software,
Northampton, MA) was used to perform least-squares fitting and to
create figures. Data are presented as mean ± S.E. or fitted
value ± S.E. of the fit. An unpaired Student's t test
(SigmaStat; Jandel Scientific Software, San Rafael, CA) was used to
evaluate the significance of changes in mean values. p
Values < .05 were considered statistically significant.
Pulse Protocols.
It was recently demonstrated that two
affinities for LA drugs can be distinguished that correspond to binding
to the resting state of Na+ channels, measured at
strongly negative potentials (
140 mV), and to the inactivated state
of Na+ channels, measured at less negative
potentials (
70 mV; Wright et al., 1997). These binding
affinities of resting and inactivated channels could be estimated
directly for LAs that unbind slowly from inactivated channels with the
following voltage pattern (Fig. 1B,
inset): a 10-s conditioning prepulse to various potentials was applied
to allow binding to reach the steady state; then, a 100-ms interval at
the holding potential of 140 mV was inserted before delivery of the
test pulse to +30 mV to allow drug-free channels to recover from fast
inactivation (Wright et al., 1997). For the LA bupivacaine, this pulse
protocol is applicable, because recovery from block is slow (e.g., see
Fig. 4). The conditioning prepulse duration of 10 s was required
to reach steady-state binding of cocaine to inactivated channels. Under
these conditions, however, a significant number of slow inactivated
channels were induced in some mutations at more positive potentials.
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Results |
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Top
Summary
Introduction
Materials and Methods
Results
Discussion
References
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State-Dependent Block of µ1 Wild-Type Na+
Channels toward Bupivacaine Enantiomers.
We applied the three-step
protocol (see Fig. 1B, inset, and Materials and Methods) in
the absence and presence of 100 µM R(+)- or S()-bupivacaine to
initially assess steady-state block of µ1 wild-type
Na+ channels. Conditioning prepulse potentials
ranged in amplitude from 160 to 50 mV. Figure 1A shows µ1
wild-type Na+ currents in the absence of drugs
and in the presence of 100 µM R(+)- and S()-bupivacaine after
conditioning prepulses to 140 or 70 mV. Both enantiomers blocked
the current to a similar degree after the prepulse to 140 mV. The
block was greater after the prepulse to 70 mV and more so for
R(+)-bupivacaine than for S()-bupivacaine.
100
mV. Block by 100 µM R(+)- and S()-bupivacaine reached plateaus at
potentials 140 and 70 mV, confirming the existence of
two distinguishable binding affinities for both bupivacaine
enantiomers that are revealed by the pulse protocol (Wright et al.,
1997).
To determine more accurately the potencies of bupivacaine enantiomers
in blocking resting and inactivated channels,
IC50 values were determined in
concentration-inhibition experiments for both enantiomers; we chose
conditioning prepulses to 140 and 70 mV, corresponding to block of
resting and inactivated channels, respectively (Fig. 1C). The
IC50 values for block by R(+)- and
S()-bupivacaine were 160 ± 21 and 146 ± 14 µM,
respectively, for resting channels and 8.6 ± 0.5 and 12 ± 1 µM, respectively, for inactivated channels. The Hill coefficients
were close to unity, which suggests a single binding site for
bupivacaine enantiomers in µ1 wild-type Na+
channels. The stereopotency ratios for R(+)- over S()-bupivacaine were significantly different between resting (0.9) and inactivated channels (1.4; p < .05). These results confirm
previous reports that Na+ channels display only
weak stereoselectivity toward bupivacaine enantiomers (Wang and Wang,
1992; Valenzuela et al., 1995).
The residue µ1-N434 in D1-S6 of µ1 wild-type
Na+ channels is asparagine (N). Asparagine has an
uncharged side chain but is decidedly polar and thus is a hydrophilic
amino acid. By using site-directed mutagenesis, we made a series of
amino acid substitutions at position µ1-N434 that vary the
hydrophobicity, aromaticity, polarity, and charge at this site. We
chose alanine (A) as a small aliphatic, hydrophobic residue; the
aromatic residues phenylalanine (F), tryptophan (W), and tyrosine (Y);
the hydroxyl- or sulfur-containing, and therefore polar, residues
threonine (T) and cysteine (C); the acidic, negatively charged residue
aspartic acid (D); and the positively charged residues lysine (K) and
arginine (R). All mutants expressed sufficient
Na+ currents in HEK 293t cells for
pharmacological analysis.
We did not aim for a detailed analysis of the gating properties of the
channels in the present study. Activation kinetics were not
measured because activation, in contrast to steady-state inactivation,
was shown to play a minor role in modulation of LA affinity (Wright et
al., 1999). However, we briefly characterized fast and slow
inactivation of µ1-N434 mutant channels.
Fast and Slow Inactivation of µ1-N434 Mutant Channels.
A
conventional two-pulse protocol was used to determine the voltage
dependence of fast inactivation (Fig. 2A,
inset). The V0.5 values for µ1-N434T and
µ1-N434R channels showed no statistically significant difference
compared with µ1 wild-type channels. The V0.5
values for µ1-N434Y, µ1-N434C, µ1-N434W, µ1-N434F, and
µ1-N434D were shifted to more negative potentials by 32.1, 16.7, 16.1, 14.2, and 5.2 mV, respectively. The V0.5
values for µ1-N434K and µ1-N434A were shifted to more positive
potentials by 4.4 and 12.6 mV, respectively (Table
1).
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100 mV began to induce slow
inactivation in µ1 wild-type channels. µ1-N434D and µ1-N434K
exhibited similar and µ1-N434R less slow inactivation. In µ1-N434Y
currents, slow inactivation was detectable at prepulses more positive
than 140 mV and in µ1-N434C, µ1-N434F, and µ1-N434W currents at
prepulses more positive than 120 mV. Slow inactivation increased
gradually with more positive potentials. In µ1-N434A and µ1-N434T,
currents slow inactivation was detectable at prepulses more positive
than 90 mV and increased steeply with more positive potentials. All
of these data were fitted by a Boltzmann equation. The midpoint
potentials S'0.5 and the corresponding slope
factors kS' for control data and data obtained in the presence of 10 or 100 µM R(+)- and S()-bupivacaine (not shown) are given in Table 1.
It is noteworthy that the S'0.5 values may not
represent the true steady-state slow inactivation because the
conditioning prepulse duration of 10 s may be too short to allow
all mutants to equilibrate to their slow inactivation state. The
S'0.5 values characterize the slow inactivation
specifically induced by the pulse protocol applied in this study to
measure steady-state block. The results confirm previous reports that a
mutation at µ1-N434 position can strongly influence both fast and
slow inactivation properties of µ1 Na+ channels
(Wang and Wang, 1997).
State-Dependent Block of µ1 Na+ Channels by
Bupivacaine Enantiomers.
For all mutant channels, steady-state
block by bupivacaine enantiomers was first assessed at different
prepulse potentials, as described above. Blocking potencies for resting
and inactivated channels were subsequently determined in
concentration-inhibition experiments with prepulses at which block
reached a plateau (that is, a point at which inhibition did not further
decrease with more negative prepulses, for resting channels, or
increase with more positive prepulses, for inactivated channels). For
wild-type and all mutant channels, a prepulse potential of 140 mV was
deemed sufficient to estimate the bupivacaine potencies for resting
channels, with the exception of µ1-N434Y and µ1-N434C, for which
160 mV was used. Potencies for inactivated channels were all
estimated with a prepulse potential of 70 mV, at which block of µ1
wild-type, µ1-N434T, µ1-N434D, µ1-N434K, and µ1-N434R clearly
approached a plateau. However, at prepulse potentials more positive
than 70 mV, block of µ1-N434A channels decreased slightly. Maximum block of µ1-N434Y, µ1-N434W, µ1-N434C, and µ1-N434F channels
was already induced at potentials of 90 and 80 mV, respectively, and decreased slightly with more positive potentials, so we may have
underestimated block of inactivated channels in these mutations.
) and/or
of the contribution of slow inactivated channels to bupivacaine block.
It was beyond the scope of the present study to determine
quantitatively the relative contribution of open and slow inactivated
states to the binding affinity at the critical potentials. When
comparing IC50 values of mutations µ1-N434Y,
µ1-N434W, µ1-N434C, and µ1-N434F obtained at a prepulse potential
of 70 mV with other mutants, however, one should keep this limitation
in mind.
The effects of 100 µM R(+)- and S()-bupivacaine on wild-type and
mutant Na+ currents elicited after prepulses to
140 and 70 mV are shown in Fig. 3A
and the IC50 values resulting from
concentration-inhibition experiments are summarized in Fig. 3, B and C. The channels are arranged from top to bottom traces (Fig. 3A) and from
left to right (Fig. 3, B and C) according to their hydropathy index
beginning with the most hydrophobic substitutions. All
IC50 values and Hill coefficients are also listed
in Table 2, along with calculated ratios
for state-selective and stereoselective potencies.
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)-bupivacaine was increased in all mutant channels. This increase was the smallest (<1.5-fold) for mutations µ1-N434K and µ1-N434R, containing positively charged residues, and moderate (about 2-fold) for
mutation µ1-N434D and µ1-N434A, containing a negatively charged or
a small hydrophobic residue, respectively. Mutations containing a polar
residue (µ1-N434T and µ1-N434C) showed a 2.5- to 6-fold increase in
blocking potency, and mutations containing an aromatic residue
(µ1-N434F, µ1-N434W, and µ1-N434Y) seemed to have the highest
sensitivity to block of both resting and inactivated states by
bupivacaine enantiomers.
In inactivated channels, the increase in potency was greater for
mutation µ1-N434D than for mutations µ1-N434C and µ1-N434T. Potency was decreased in mutation µ1-N434K. The potency to block inactivated µ1-N434R channels decreased for S()-bupivacaine but remained constant for R(+)-bupivacaine, resulting in a
stereoselectivity (stereopotency ratio) of 3 for R(+)- over
S()-bupivacaine (Table 2) for the inactivated state of µ1-N434R channels.
Recovery of Inactivated µ1-N434R Channels from Block by
Bupivacaine Enantiomers.
Different rates of dissociation of R(+)-
and S()-bupivacaine from inactivated µ1-N434R
Na+ channels during the interval at 140 mV
before the test pulse may confound the estimation of block of
inactivated channels at 70 mV. Therefore, we determined the
percentage of block of inactivated µ1 wild-type and µ1-N434R
channels and their recovery time course from block by bupivacaine
enantiomers (Fig. 4).
|
)-bupivacaine recovered with fast (
1)
and slow time constants (2). In the presence
of a drug, 1 reflects the fast recovery from
inactivation of unblocked channels; 2 reflects the slow dissociation of drug from channels that were blocked during
the conditioning prepulse, including rebinding and dissociation from
resting channels and slow recovery from inactivation seen also under
control conditions. Control currents of mutation µ1-N434R (Fig. 4B)
recovered from a 10-s prepulse to 70 mV with fast and slow time
constants of 4.6 ± 0.9 ms and 1.1 ± 0.2 s,
respectively. In the presence of 100 µM R(+)-bupivacaine, these
channels recovered with fast and slow time constants of 6.2 ± 0.2 ms and 2.8 ± 0.1 s, respectively, and in 100 µM
S()-bupivacaine with respective values of 4.7 ± 1.2 ms and
1.7 ± 0.1 s. The fractional amplitudes of the slow phase of
recovery for R(+)- and S()-bupivacaine were 92 and 73%,
respectively, resulting from the different potencies of bupivacaine
enantiomers to block inactivated µ1-N434R channels. The slow time
constant for recovery from R(+)-bupivacaine block was 1.6-fold longer
than that for S()-bupivacaine. More importantly, the results clearly
show that at 100-ms recovery time, little recovery of inactivated
drug-bound channels had occurred, thus confirming the significant
difference in block by R(+)- and S()-bupivacaine and validating the
pulse protocol used to estimate block of inactivated µ1-N434R channels.
Because of the large fraction of inactivated channels blocked by either
100 µM R(+)- or S()-bupivacaine (>90%), no reliable fast time
constants could be estimated for µ1 wild-type channels (Fig. 4A).
Dissociation from blocked µ1 wild-type channels was slower for both
R(+)- and S()-bupivacaine (4.4 ± 0.1 and 2.3 ± 0.1 s, respectively) than the corresponding dissociation from µ1-N434R channels. It is noteworthy that the recovery from block by
R(+)-bupivacaine was about two times slower than from block by
S()-bupivacaine, although stereoselectivity was weak for both resting
and inactivated µ1 wild-type channels, revealing that stereoselective
actions are not determined by differences in dissociation kinetics alone.
Block of µ1-N434R Na+ Channels by Other LA
Stereoisomers.
We were interested in whether the increased
stereoselectivity for bupivacaine of inactivated µ1-N434R channels
also occured with other LA stereoisomers. Therefore, we assessed
steady-state block of µ1 wild-type and µ1-N434R channels by (+)-
and ()-cocaine and by the enantiomers of the LA compound RAC 109. All
IC50 values and Hill coefficients for block by
cocaine-enantiomers are listed in Table
3.
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)-cocaine
over (+)-cocaine (Table 3). Hence, the differential reduction in
blocking potency of inactivated µ1-N434R channels toward one
stereoisomer is consistent for both bupivacaine and cocaine.
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Discussion |
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Top
Summary
Introduction
Materials and Methods
Results
Discussion
References
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Our data demonstrate that most point mutations at µ1-N434 had significant effects on both fast and slow inactivation of Na+ currents and on potencies of bupivacaine enantiomers to block resting and inactivated channels. In the following, we equate the term binding affinity with blocking potency measured in concentration-inhibition experiments when appropriate. We thereby assume that block of Na+ channels by LAs measured in electrophysiological experiments directly reflects binding of LAs to their site.
Changes in Fast and Slow Inactivation.
Mutations µ1-N434Y,
µ1-N434F, µ1-N434W, µ1-N434C, and µ1-N434A had the most
dramatic effects on both fast and slow inactivation. All of them
enhanced slow inactivation, as shown in the leftward shift of the
S'
curve. However, µ1-N434Y, µ1-N434W,
µ1-N434F, and µ1-N434C shifted the voltage dependence of fast
inactivation (h) to the hyperpolarizing
direction, whereas µ1-N434A shifted it to the depolarizing direction.
These data together demonstrate that fast and slow inactivation can be
differentially modulated through substitution of residue N434.
; West
et al., 1992) and amino acid residues near the intracellular end of
D4-S6 (McPhee et al., 1994) and within the short intracellular loop
between D4-S4 and D4-S5 (McPhee et al., 1998). However, numerous
mutations in other regions of the Na+ channel
protein are known to modulate fast inactivation as well.
Less is known about the molecular structures that govern slow
inactivation of Na+ channels, although there is
growing evidence for an important role of segments S6. Enhancement
of slow inactivation (Fig. 2B), together with positive shifts in
the voltage dependence of activation and fast inactivation in mutation
µ1-N434A in D1-S6, has been described (Wang and Wang, 1997). A
mutation of an external pore-flanking residue in D1-S6 eliminated the
anomalous slow recovery from inactivation of the subunit of µ1
Na+ channels expressed in X. laevis
oocytes (Balser et al., 1996a). Both reports point out the functional
and structural analogy of slow inactivation in
Na+ channels to the C-type inactivation in
homotetrameric Shaker K+ channels that
is modulated by external pore-lining residues and by residues in
segment S6 (Hoshi et al., 1991; Boland et al., 1994; Yellen et al.,
1994). Interestingly, segment S6 in Shaker K+ channels may be involved in the
activation-gating mechanism as well (Liu et al., 1997; Holmgren et al.,
1998). Whether segment D1-S6 of the Na+ channel
plays a comparable role in channel gating remains unanswered.
Changes in Bupivacaine Affinity. As in the wild-type, two distinguishable bupivacaine binding affinities corresponding to the resting and inactivated states were found in all mutant channels. Mutations of N434 clearly affected binding of LAs to resting and inactivated channels differently.
In theory, three possible mechanisms may explain altered LA affinities: 1) mutations at N434 alter the gating properties and consequently affect state-dependent binding; (2) mutations at N434 influence LA binding by indirect allosteric effects at the site; or 3) residues at N434 directly interact with LA in binding to the Na+ channel. Mutations at N434 indeed had marked effects on gating properties of the channels. However, shifts in the voltage dependence of steady-state inactivation elicited linear shifts in the voltage dependence of steady-state block but did not affect LA affinity of resting and inactivated channels (Wright et al., 1999). In the present study,
affinities of resting and inactivated channels toward LAs were
determined outside the potential range (120 to 80 mV) where
resting-to-inactivated transitions occur in the presence of bupivacaine
enantiomers and should not be influenced by shifts in the voltage
dependence of fast inactivation.
Affinities of inactivated channels were estimated with conditioning
prepulses to 70 mV, a potential that induced considerably different
percentages of slow inactivated channels in the mutants. Consequently,
slow-inactivated channels may contribute to LA block. Slow-inactivated
states of µ1 Na+ channel subunits expressed
in X. laevis oocytes were shown to have lidocaine affinity
comparable with that of fast-inactivated states in
1 coexpressed channels (Balser et al.,
1996b). It is unclear, however, whether the slow-inactivated states
observed in oocytes and in mammalian expression systems are comparable.
Ultimately, based on our results, we could not discern a clear
relationship between shifts in fast or slow inactivation in mutant
channels and their resting and inactivated affinities toward bupivacaine (Tables 1 and 2). Altogether, it seems that the altered LA
binding affinities are not caused by the altered gating properties in
mutant channels.
At present, we cannot exclude allosteric effects at the LA site caused
by mutations in N434 as an explanation for changes in affinity.
However, there is compelling structure-function evidence for a direct
interaction of N434 with bupivacaine enantiomers, as is most
apparent in changes in the affinity of inactivated mutant channels.
Bupivacaine affinity of inactivated channels was enhanced for mutations
in the order µ1-N434Y, µ1-N434F, µ1-N434W, µ1-N434D, and
µ1-N434C, whereas it was decreased for mutations µ1-N434K and
µ1-N434R. The most likely explanation is that these residues interact
directly with the positively charged amino group of bupivacaine. The
aromatic residues phenylalanine, tryptophan, and tyrosine could
increase affinity through cation- electron interaction (Heginbotham
and MacKinnon, 1992), as suggested for the rat brain IIA native residue
F1579 and the positively charged moiety of an LA (Ragsdale et al.,
1994). The negatively charged aspartic acid and the polar cysteine may
interact electrostatically with the positively charged LA moiety to
increase affinity. The positively charged lysine and arginine decrease
binding of the positively charged LA moiety by an electrostatic
charge-charge repulsion. This mechanism also explains the reduced
inactivated channel block by LAs in lysine point mutations at the
putative LA binding site in D4-S6 (Wright et al., 1998). Consistent
with a direct interaction of residues at N434 with a positively charged LA moiety is the unchanged bupivacaine affinity of inactivated channels
in mutation µ1-N434A, where asparagine is replaced by the small
hydrophobic alanine, and the small increase in the affinity in mutation
µ1-N434T, where asparagine is replaced by the slightly polar threonine.
Surprisingly, all resting mutant channels showed a higher bupivacaine
affinity than the resting wild-type channels. The changes in
blocking potencies were not well correlated with the physical properties of the substituted residues.
These results might be interpreted like those regarding the
phenylalanine residue in D4-S6 known to be critical for LA action, which appears to interact with LA in two different modes depending on
the channel's state: in the resting state, hydrophobic interactions are more important for LA binding, whereas in the open and inactivated states, cation- electron or aromatic-aromatic interactions are dominant (Li et al., 1999). The speculation that the orientation of the
residue's side chain changes in response to channel state could also
apply to our results. Alternatively, mutations in N434 may
nonspecifically affect LA binding to resting channels.
The role of residue N434 in LA binding to resting channels remains
elusive. Nonetheless, different forces seem to regulate LA binding to
resting and inactivated channels, thus suggesting that during state
transitions, the LA receptor indeed alters its configuration, as
implied by Hille's modulated receptor hypothesis (Hille, 1977).
What Is the Mechanism for the Altered Stereoselectivity by
N434R?
The most surprising result in this study was the increased
stereoselectivity of inactivated µ1-N434R channels toward bupivacaine enantiomers. Resting µ1-N434R channels showed little
stereoselectivity. Stereoselectivity for the inactivated state arose
from a decrease in affinity for S()-bupivacaine with no change for
R(+)-bupivacaine. Mutation µ1-N434K introduces the same positive
charge, albeit of smaller dimension, yet shows no stereoselectivity.
the amine-containing butyl piperidine ringmay be near one
another, consistent with the proposed direct interaction. Second,
R(+)-bupivacaine remains bound to inactivated µ1-N434R channels with
minimal charge-charge repulsion, possibly because of delocalized charge
in the guanidinium group of the arginine residue. Third, in addition
to charge, the size and the orientation of atoms and bonds
within the residue at 434 position are crucial for enhanced stereoselectivity.
The differential reduction in affinity of inactivated µ1-N434R
channels toward one bupivacaine stereoisomer was also found for
cocaine, which suggests that corresponding structural parts of
bupivacaine and cocaine interact with the same residues. In both
bupivacaine and cocaine molecules, the chiral carbon is close to the
tertiary amine. In contrast, the chiral carbon of RAC 109 is located
close to the aromatic part of the molecule, the moiety that might not
interact with the residue at position µ1-N434. Additionally, the
bulkier structure of RAC 109 may cause steric hindrance in binding to
resting and inactivated µ1-N434R channels.
In conclusion, we propose that in inactivated channels, residue
µ1-N434 in D1-S6 interacts directly with the positively charged moiety of the LAs bupivacaine and cocaine. Our findings provide more
evidence that segments D1-S6 and D4-S6 align adjacently along the pore
of the Na+ channel, forming a domain-interface
site for binding of BTX and of LAs in close proximity.
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Acknowledgments |
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We thank to Dr. Stephen Cannon for providing the HEK 293t cell line and the CD8-pih3 m plasmid and Dr. James Trimmer for providing the µ1/skm1 plasmid.
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Footnotes |
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Received December 31, 1998; Accepted April 30, 1999
This study was supported by National Institutes of Health Grants GM35401 and GM48090 (to G.K.W and S.-Y.W.) and by a stipend of the Deutsche Forschungsgemeinschaft (to C.N).
Send reprint requests to: Dr. Carla Nau, Department of Anesthesia Research Laboratories, Brigham and Women's Hospital, 75 Francis St., Boston, MA 02115. E-mail: cnau{at}zeus.bwh.harvard.edu
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Abbreviations |
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LA, local anesthetic; BTX, batrachotoxin; HEK, human embryonic kidney.
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References |
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Top
Summary
Introduction
Materials and Methods
Results
Discussion
References
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subunit.
J Biol Chem
271:
11261-112671 subunit.
Biophys J
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) were normalized to
the current obtained with a prepulse to 
) and S(
) were combined.
Currents obtained in the presence of 100 µM R(+)- or
S(
) and
(+)-cocaine (
), and (C) 100 µM RAC 109 I (
) and RAC 109 II
(
). Data were obtained as described in Fig.