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Vol. 54, Issue 4, 733-739, October 1998
Department of Anesthesia Research Laboratories, Harvard Medical School, Brigham and Women's Hospital, Boston, Massachusetts 02115 (S.N.W., G.K.W.), and Department of Biology, State University of New York at Albany, Albany, New York 12222 (S.-Y.W.)
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Summary |
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Top
Summary
Introduction
Materials & Methods
Results
Discussion
References
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Voltage-gated Na+ channels are a primary target for local
anesthetics (LAs). Open or inactivated Na+ channels usually
have a severalfold higher affinity for LAs than do resting channels.
Hille's modulated receptor hypothesis attributed the changes in LA
affinity to state-dependent alterations in the conformation of the LA
receptor. We expressed wild-type and mutant rat skeletal muscle (µ1)
Na+ channels in human embryonic kidney cells to investigate
the state-dependent modulation of LA receptor affinity. As an
alternative approach to using alanine for point mutation, we
substituted lysine (a hydrophilic residue) for native residues in the
putative LA receptor located in D4-S6 of the µ1 Na+
channel. Lysine mutation at Y1586 did not alter resting channel affinity for cocaine but did reduce resting affinity at F1579K and
N1584K by 2- and 3-fold, respectively. Compared with µ1, resting benzocaine block did not change at F1579K, decreased at N1584K, and
increased at Y1586K. These effects on resting block could largely be
accounted for by either steric/charge interference or cation-
electron interactions between particular moieties on the LA and lysine.
Surprisingly, lysine substitution at these residues allowed the
channels to undergo steady state fast inactivation yet reduced
inactivated channel block by cocaine by up to 27-fold and reduced the
benzocaine-induced leftward shift in the h
curve by up
to 22 mV. Our data suggest that transitions in channel state indeed
invoke conformational changes in the LA receptor and that lysine
mutations in the LA receptor region alter such conformational changes
during the transition to the inactivated state.
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Introduction |
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Top
Summary
Introduction
Materials & Methods
Results
Discussion
References
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Voltage-gated
Na+ channels are membrane proteins that produce
action potentials in excitable tissues. The 
subunit of
Na+ channels consists of four homologous domains,
each of which contains six transmembrane segments that supposedly have
an -helical secondary structure (Numa and Noda, 1986
). LA agents
block the transmission of action potentials by binding to a receptor
site on the Na+ channel subunit. According to
the modulated receptor model for LAs (Hille, 1977), channel state
governs the conformation of the LA receptor. The receptor has a weak
affinity for LA in the resting state but a strong affinity for LA in
the activated or inactivated state. Ragsdale et al. (1994)
first showed that point mutation of specific residues to alanine in
Domain 4-S6 of the rat brain Na+ channel (NaIIa)
strongly affected LA block of Na+ currents when
the channels were expressed in Xenopus laevis oocytes. Recently, Wang et al. (1998) reported similar findings for
comparable mutations of the rat skeletal muscle
Na+ channel (µ1; Trimmer et al.,
1989) when expressed in mammalian cells (HEK 293T). The study by
Wang et al. (1998) also confirmed that etidocaine, a
tertiary amine LA, and neutral benzocaine (ethyl p-aminobenzoate) bind to a common receptor within Domain
4-S6.
In the present study, we substituted a charged amino acid (lysine) for
single point mutations of the native residues at µ1-F1579, N1584, and
Y1586 and expressed the channels in HEK cells. Point mutation of
comparable residues in NaIIa channels to alanine (NaIIa-F1764A, N1769A,
and Y1771A) markedly altered receptor affinity for etidocaine (Ragsdale
et al., 1994). Our aim was to determine how incorporation of
a charged residue at these positions might affect state-dependent LA
affinity. We examined block of Na+ current by two
LAs that are structurally very different. Cocaine (a tertiary amine)
has a large rigid structure and the charged species, presumed to be
active at the receptor (Narahashi et al., 1970; Nettleton
and Wang, 1990), is predominant at neutral pH. Benzocaine, on the other
hand, is a more flexible compound and is uncharged. Our results
indicated that lysine mutation at these three residues had little
effect on the level of current expression and nearly eliminated high
affinity, inactivated channel binding of both cocaine and benzocaine.
The reduction in inactivated channel block could not be attributed to
changes in channel gating because the mutations did not prevent the
mutant channels from entering the fast inactivated state. Our data
suggest that lysine mutation of residues within the LA receptor region
prevents the receptor from occupying the high affinity
conformation without drastically altering the kinetic transition from
resting to inactivated channels.
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Materials and Methods |
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Top
Summary
Introduction
Materials & Methods
Results
Discussion
References
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Solutions and chemicals. Cocaine hydrochloride was purchased from Mallinckrodt (St. Louis, MO), and benzocaine was purchased from Sigma Chemical (St. Louis, MO). LAs were applied externally at the appropriate concentration. The extracellular solution used to perfuse HEK cells contained: 65 mM NaCl, 85 mM choline Cl, 2 mM CaCl2, and 10 mM HEPES (titrated with tetramethylammonium hydroxide to pH 7.4). The pipette solution contained 100 mM NaF, 30 mM NaCl, 10 mM EGTA, and 10 mM HEPES (titrated with cesium hydroxide to pH 7.2).
Mutagenesis of µ1 channels and transient transfection of HEK
293T cells.
Site-directed mutagenesis was used to create lysine
point mutations of the µ1-pcDNA/amp vector at residues µ1-F1579,
µ1-N1584, and µ1Y1586 as described previously (Wang and Wang, 1998;
Wang et al., 1998). One additional application of nucleoside
triphosphates and T4-DNA polymerase was given during the 4-hr in
vitro synthesis. Potential mutants were identified by restriction
mapping and confirmed by DNA sequencing.
), HEK 293T
cells were transfected with channel plasmid (2-10 µg) and reporter plasmid CD8-pih3m (1 µg) by the calcium phosphate precipitation method (Cannon and Strittmatter, 1993). The transfected cells were
replated onto 35-mm culture dishes and used for experiments for
up to 3 days. Transfection-positive cells, as identified by CD8
Dynabeads (Dynal, Lake Success, NY), were selected for whole-cell patch
recording. Expression levels of the lysine mutants were generally > 1 nA, which were equal to or greater than the expression levels of corresponding alanine-substituted mutants (Wang et
al., 1998).
Electrophysiology and data analysis.
Whole-cell voltage
clamp (Hamill et al., 1981) of HEK cells was used to study
macroscopic Na+ currents at room temperature
(23 ± 2°). Electrode resistances ranged from 0.4 to 1.0 M
.
Command voltages were programmed by pCLAMP software (Axon Instruments,
Burlingame, CA) and delivered by a List EPC7 voltage clamp. Data were
sampled at 50 kHz and filtered at 5 kHz. After establishment of
whole-cell voltage clamp, the cells were dialyzed for 25-30 min before
data were acquired (Wright et al., 1997). The holding
potential for all experiments was 
140 mV. Most of the capacitative
current was canceled by the EPC7 circuitry; any remaining capacitative
artifact and the leakage current were subtracted by the P/4 method.
The voltage error at +30 mV was 
5 mV. Pulses were separated by 5 sec
when acquiring steady state activation and inactivation data (including steady state inactivation in benzocaine). For all other experiments, pulses were separated by 30 sec intervals at the holding potential. Least-squares curve-fitting was performed with Origin software (Microcal, Northampton, MA). Statistical analyses (Student's
t test) were performed using SigmaStat (Jandel Scientific
Software, San Rafael, CA) to determine the significance of changes in
mean values. p values of < 0.05 were considered
statistically significant; n values indicate the number of
cells examined. Data are presented as mean ± standard error
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Results |
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Top
Summary
Introduction
Materials & Methods
Results
Discussion
References
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Effects of lysine point mutations on µ1 channel kinetics.
To
determine the voltage dependence of activation of µ1 and mutant
channels, we measured the peak Na+ current during
5-msec depolarizations to test potentials ranging from 100 mV to +50
mV (Fig. 1A). Activation of N1584K
channels closely resembled the activation of µ1 channels, whereas the
midpoint voltages (V0.5) of activation for F1579K
and Y1586K were 11 mV and 12 mV, respectively, more positive than the
V0.5 value of activation for µ1. To determine
the voltage dependence of steady state inactivation
(h) of the channels, we delivered 100-msec conditioning pulses ranging from 160 mV to 35 mV and measured the
peak Na+ current during a 5-msec test pulse to
+30 mV (Fig. 1B). The V0.5 value of steady state
inactivation for Y1586K channels was similar to that of µ1 channels,
whereas V0.5 value of steady state inactivation for F1579K and N1584K channels were 6 mV and 12 mV, respectively, more
positive than that of µ1. In addition, approximately 10% of the
N1584K channels did not inactivate after conditioning pulses ranging
from 50 mV to 35 mV.
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Lysine point mutations in Domain IV-S6 affect state-dependent
modulation of cocaine affinity.
To obtain an initial assessment of
cocaine sensitivity after lysine point mutation, we examined cocaine
block of the channels using a pulse protocol that consisted of 10-sec
conditioning pulses to various voltages followed by a 100-msec interval
at the holding potential and a subsequent test pulse to +30 mV (Fig.
2). The 10-sec conditioning pulses
allowed cocaine binding to reach steady state, and the 100-msec
interval at the holding potential permitted inactivated but drug-free
channels to recover from fast inactivation (Wright et al.,
1997). The percentage of block after strongly negative conditioning
pulses provided an estimate of resting channel affinity for cocaine
whereas the percentage of block after the least negative conditioning
pulses provided an estimate of the inactivated channel affinity.
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140 mV (Fig. 2A,
left traces) and to 70 mV (Fig. 2A, right
traces). After a conditioning pulse to 140 mV, 50 µM cocaine blocked similar percentages of current at
resting µ1 and Y1586K channels but blocked a smaller percentage of
current at resting F1579K and N1584K channels. After inactivating the
channels with a 10-sec conditioning pulse to 70 mV, 50 µM cocaine blocked a much larger percentage of µ1 current than at any of the three mutant channels. Although the conditioning pulse to 70 mV in control saline elicited large amounts
of slow inactivation at Y1586K channels (Fig. 2A, dashed line), the cocaine-induced reduction in test-current amplitude was
much less at Y1586K channels than at µ1 channels.
Fig. 2B shows the effect of the pulse protocol on normalized test
currents evoked in control saline. We normalized the control data by
dividing the peak amplitude of the test current at each conditioning
voltage by the peak amplitude of the test current evoked after the
conditioning pulse to 160 mV. In control saline, 10-sec conditioning
pulses more negative than 80 mV had little effect on the test current
amplitude of µ1, F1579K, or N1584K channels. Conditioning pulses more
positive than 80 mV, which began to elicit slow inactivation, had
similar effects on test current amplitude at µ1 and F1579K channels
but had little effect on the current amplitude at N1584K channels. In
contrast, conditioning pulses more positive than 120 mV elicited
increasing amounts of slow inactivation at Y1586K channels.
We delivered the pulse protocol in the presence of cocaine (Fig. 2C) to
determine the cocaine sensitivities of resting and inactivated
channels. To obtain the percentage of cocaine block at each
conditioning voltage, we divided the peak amplitude of the test
currents evoked in 50 µM cocaine by the peak amplitude of
the test currents evoked in control saline. Cocaine block of the
resting channels (160 mV to 120 mV) was weak, although both F1579K
and N1584K channels seemed less sensitive than either µ1 or Y1586K.
As the conditioning voltage became less negative, an increasing
percentage of µ1 channels became fast inactivated and subsequently
blocked by cocaine during the 10-sec conditioning pulse. The percentage
of block of inactivated µ1 channels reached steady state at 70 mV.
In contrast, even the most positive conditioning pulses induced very
little block of inactivated F1579K and N1584K channels. Cocaine block
of Y1586K channels resembled the block of µ1 channels up to 100 mV,
and conditioning pulses to between 90 mV and 70 mV produced modest
increases in the block of Y1586K channels. Note that after a
conditioning pulse to 60 mV (Fig. 2C), block of Y1586 decreased by
20% (p < 0.05; n = 6)
compared with block after a conditioning pulse to 70 mV. The decrease in cocaine block of Y1586K channels at 60 mV most likely resulted from modest amounts of channel activation and knockout of the drug by
external Na+ ions (Wang, 1988). These data
indicated that introduction of a lysine residue in the region of the LA
receptor markedly reduced the affinity of inactivated channels for
cocaine.
To more accurately determine the affinities of resting and inactivated
channels for cocaine, we performed dose-response experiments and fitted
the data with the Hill equation to estimate the dissociation constants
of resting (KR) and inactivated
(KI) channels (Fig. 3). In all cases, the fitted curves had a
Hill coefficient of 1, which indicates that one cocaine molecule binds
to each channel. We determined the resting channel affinity by
performing the experiments at a holding potential of 140 mV and
measuring the percentage of cocaine block at +30 mV (Fig. 3A). Cocaine
had a similar affinity for resting µ1 and Y1586K channels with
KR values of 222.5 ± 15.7 µM (n = 4) and
226.4 ± 8.6 µM (n = 6; p > 0.05), respectively. Compared with µ1,
resting F1579K and N1584K channels had a 2- to 3-fold
(p < 0.05) lower affinity for cocaine with
KR values of 455.5 ± 23.4 µM (n = 4) and
631.1 ± 33.1 µM (n = 7), respectively (Table 1).
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140 mV and a subsequent test pulse to +30 mV (Fig. 3B). We used a
conditioning pulse to 70 mV for determining the inactivated channel
affinities of µ1, F1579K, and Y1586K. Because the
V0.5 value of steady state inactivation for
N1584K channels was about 10 mV more positive than the
V0.5 values for the other channels (Fig. 1B), we
determined the inactivated channel affinity of N1584K channels using a
conditioning pulse to 60 mV. Lysine point mutation substantially
reduced the relative increase in cocaine affinity at inactivated
channels
(KR/KI
ratio; Table 1). At µ1 channels, the
KR value for cocaine was 18 times
larger than the KI value. In
contrast, the
KR/KI
ratios at F1579K, N1584K, and Y1586K channels were 1.8, 1.9, and 3.1, respectively. Furthermore, lysine point mutation resulted in a markedly
reduced inactivated channel affinity compared with the inactivated
channel affinity of µ1 channels. The reduction in inactivated channel
affinity was smallest at Y1586K channels (~6-fold) and was largest at
N1584K channels (~27-fold). Note that inactivated F1579K channels had a similar affinity for cocaine, and inactivated N1584K channels had a
weaker affinity for cocaine compared with µ1 resting affinity at
140 mV.
Effect of lysine point mutation on block by neutral
benzocaine.
To estimate the benzocaine affinity of resting
channels, we measured the percentage of current blocked by 1 mM benzocaine during a step from the holding potential to
+30 mV (Fig. 4, A and B). Interestingly,
benzocaine block of resting channels differed at each of the three
mutant channels. On average, 1 mM benzocaine blocked a
similar amount of current at µ1 channels (40%) and F1579K channels
(41%; p > 0.05). Compared with block of µ1
channels, 1 mM benzocaine blocked a significantly smaller
(p < 0.05) percentage of current at N1584K
channels (35%) but a significantly larger (p < 0.05) percentage of current at Y1586K channels (55%). We did not
perform dose-response experiments with benzocaine because the Hill
coefficient varies from < 1 to > 1 depending on the
concentration range (Meeder and Ulbricht, 1987; Wang et
al.,. 1998) and because the solubility of benzocaine is less than
4 mM.
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curves
(Fig. 4C) induced by 1 mM benzocaine. We assumed that the
magnitude of the leftward shift provided relative information about the affinity of benzocaine at inactivated channels. Benzocaine shifted the
h curve of µ1 channels by 26 mV in the
negative direction, whereas the negative shifts at F1579K, N1584K, and
Y1586K channels were 7 mV, 4 mV, and 18 mV, respectively. For each
channel, the effect of benzocaine on the Boltzmann function slope
factor (k) reflected the magnitude of the leftward shift,
with µ1 having the largest increase in k value (see
legend, Fig. 4C). Thus, the reduction in leftward shift in
h at the three mutant channels was generally
consistent with the reduction in cocaine affinity for the inactivated
state. For both cocaine and benzocaine, the affinity of inactivated
N1584K channels was most reduced and the affinity of inactivated Y1586K
channels was least reduced.
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Discussion |
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Top
Summary
Introduction
Materials & Methods
Results
Discussion
References
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The idea that channel state modulates the conformation and
affinity of the LA receptor (Hille, 1977) has been well supported (for
review, see Butterworth and Strichartz, 1990). The inherent complexity
of a state-dependent modulated receptor and the lack of information
about the receptor's conformational changes that accompany channel
state transition have thus far hampered the study of LA action. Recent
studies have shown that residues within Domain 4-S6 of rat brain IIa
Na+ channels are important determinants for
binding etidocaine (Ragsdale et al., 1994) as well as Class
I antiarrhythmic drugs and anticonvulsant agents (Ragsdale et
al., 1996). In this study, we explored the mechanism of
state-dependent receptor modulation using lysine point mutations of
critical residues within Domain 4-S6 of µ1 Na+
channels. Our data generally support the LA binding model described by
Ragsdale et al. (1994) and offer further explanation into
the interaction between LA agents and voltage-gated
Na+ channels. The major finding in this report is
that lysine mutation of the native residues at µ1-F1579, N1584, and
Y1586 nearly eliminates high affinity LA binding with the inactivated
state. The implications of our findings are that 1) these residues are
critical for LA binding and 2) lysine mutation of these residues
prevents the LA receptor from occupying the high affinity conformation
through charge interference or some unidentified allosteric mechanism.
With respect to the resting channel affinity for LA, most of our
results can be accounted for by the Ragsdale et al. (1994) model for LA binding to Na+ channels (Fig.
5A). The model contends that the contact
point for the charged region of a tertiary amine LA lies deep within the channel pore at µ1-F1579, whereas the contact point for the phenyl group lies closer to the cytoplasmic mouth of the pore at
µ1-Y1586. The attraction for LA is thought to occur through cation- electron interaction (Heginbotham and MacKinnon, 1992) between the aromatic moiety on the F1579 residue and the tertiary amine
moiety on the LA, and through hydrophobic interaction (Butterworth and
Strichartz, 1990) between the aromatic moieties on the Y1586 residue
and on the LA. Our data show that substitution of lysine at µ1-F1579
reduced resting affinity for cocaine by 2-fold (Fig. 3A and Table 1),
which suggests that the charged lysine inhibited cocaine binding with
the tertiary amine moiety, perhaps by a charge-charge interaction. On
the other hand, lysine substitution at residue µ1-Y1586 had no effect
on the resting affinity for cocaine. Replacement of the hydrophobic Y
residue with the basic lysine residue could invoke a cation-
electron interaction between the aromatic ring on cocaine and the
charged amine moiety on lysine, resulting in no net change in resting
affinity.
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We can draw similar conclusions about the effects of lysine
substitution on resting benzocaine affinity. Compared with the wild-type channels, mutation of the F1579 residue to lysine had no
detectable effect on benzocaine block of resting channels, whereas
lysine substitution at Y1586 increased block of resting channels (Fig.
4B). These results are also consistent with the Ragsdale et
al. model, because there is no tertiary amine moiety on benzocaine
to interact with residue F1579. Furthermore, the increase in benzocaine
affinity at Y1586K can be explained if a cation- electron
interaction between the aromatic ring on benzocaine and the amine
moiety on Y1586K is stronger than the hydrophobic interaction between
the aromatic ring on benzocaine and the aromatic ring on tyrosine at
Y1586. Note also that benzocaine contains a 4-amino group on its phenyl
ring that is not present on cocaine.
One significant difference between our data and those of Ragsdale
et al. (1994) was the effect on LA affinity after point mutation at residue µ1-N1584 (NaIIa-N1769). Both Ragsdale et
al. (1994) and Wang et al. (1998) showed that alanine
substitution at this residue increased the resting affinity for
etidocaine by severalfold. In contrast, substitution of lysine for
µ1-N1584 reduced receptor affinity for cocaine and benzocaine at both
resting and inactivated channels more so than did the mutations at
µ1-F1579 and Y1586. Indeed, mutation µ1-N1584K reduced cocaine
affinity at resting channels by 3-fold and reduced the inactivated
affinity by almost 30-fold. Ragsdale et al. (1994)
attributed the increase in resting etidocaine affinity at mutant
NaIIa-N1769A (µ1-N1584) to indirect effects because the model has the
residue facing away from the channel pore as shown in the helical wheel
plot of the D4-S6 segment in Fig. 5B, where the dashed line
indicates the pore lining. If alanine point mutation at µ1-N1584
increases LA affinity through indirect effects, then it is
conceivable that lysine point mutation could have vastly different
indirect effects than those of alanine. The decreases in cocaine
affinity at µ1-N1584K can not be attributed to changes in gating
because we determined KR at 140 mV
where steady state inactivation is completely removed, and we
determined KI at 60 mV where steady
state inactivation of µ1-N1584K was comparable with that of µ1 at
70 mV. Note that although approximately 10% of the N1584K channels
did not fast inactivate, this small component could not be responsible
for the cocaine resistance of the inactivated state in N1584K channels because both F1579K and Y1586K were cocaine-resistant, even though their steady state inactivation reached completion. These data imply
that residue N1584 and/or the surrounding microenvironment have a
substantial role in determining LA affinity. Further study of N1584
with hydrophilic and hydrophobic residues as substitutes may help
clarify the role of this residue in LA binding.
Our most striking observation was that lysine substitution virtually
eliminated inactivated channel block at F1579 and N1584 and markedly
reduced inactivated channel block at Y1586. In addition to the
severalfold reduction in the inactivated channel affinity for cocaine,
lysine mutation at these residues significantly decreased the
benzocaine-induced left shift in the h curve
despite having variable effects on resting benzocaine affinity. One
interpretation of our data is that lysine point mutation of the D4-S6
segment could, in theory, alter LA access to the receptor when the
channel occupies the inactivated conformation. Although we can not
completely rule out this possibility, benzocaine is a small, flexible,
hydrophobic LA whose access to the receptor would not likely be
affected by lysine point mutation. Furthermore, lysine substitution
reduced inactivated channel block by cocaine in a manner consistent
with the reduction in benzocaine block, suggesting that the mechanism for the reduction in inactivated channel block by these two very different LAs is at the level of the receptor and not a change in LA
access.
Lysine substitution at F1579 had no detectable effect on resting
benzocaine affinity but reduced the magnitude of the leftward shift in
the h curve by 20 mV. According to the
Ragsdale et al. model, F1579 should not interact directly
with benzocaine because benzocaine lacks the tertiary amine, yet
mutation of F1579 to lysine reduced the inactivated channel affinity
for benzocaine. The reduction in inactivated channel binding of
benzocaine at F1579K may be caused by charge interference or steric
interaction between the amino group of lysine and the amino group of
benzocaine when the channel occupies the high affinity conformation.
Our findings thus suggest that conformational changes in the LA
receptor indeed accompany changes in kinetic state. Furthermore, lysine mutation in the LA receptor region affected the local conformational transition of the receptor from the low affinity state to the high
affinity state without markedly affecting the global transition from
the resting state to the inactivated state. In normal channels, the
transition from the resting state to the inactivated state could
increase the affinity of the LA receptor by shifting the orientation of
the S6 segment residues. The entire helical structure of the S6
segment could twist or become tilted in response to outward movement of
the S4 segment during depolarization (Yang and Horn, 1995; Yang
et al., 1996). A more drastic alteration in receptor
configuration could occur if depolarization induces changes in the
secondary structure of the S6 segment as has been suggested for the S4
segment of K channels (Aggarwal and MacKinnon, 1996). Because the
Na+ channel selectivity filter also strongly
influences LA binding (Sunami et al., 1997), further
examination of the pore region using charged residue substitutions
should extend our present understanding of LA interactions and could
also prove to be a valuable tool for investigating the structural
determinants of other transmembrane segments of ion channels.
| |
Footnotes |
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Received May 22, 1998; Accepted June 24, 1998
This study was supported by National Institutes of Health National Research Service Award GM18760 (S.N.W.) and by National Institutes of Health Grants GM35401 and GM48090 (S.-Y.W. and G.K.W.).
Send reprint requests to: Dr. Sterling N. Wright, Department of Biological Sciences, Murray State University, P.O. Box 9, Murray, KY 42071. E-mail: sterling.wright{at}murraystate.edu
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Abbreviations |
|---|
LA, local anesthetic;
EGTA, ethylene
glycol bis(
-aminoethyl
ether)-N,N,N',N'-tetraacetic
acid;
HEK, human embryonic kidney;
HEPES, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid;
h, steady state availability function.
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References |
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Top
Summary
Introduction
Materials & Methods
Results
Discussion
References
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C. Nau, S.-Y. Wang, and G. K. Wang Point Mutations at L1280 in Nav1.4 Channel D3-S6 Modulate Binding Affinity and Stereoselectivity of Bupivacaine Enantiomers Mol. Pharmacol., June 1, 2003; 63(6): 1398 - 1406. [Abstract] [Full Text] [PDF]
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 K. Hilber, W. Sandtner, O. Kudlacek, B. Schreiner, I. Glaaser, W. Schutz, H. A. Fozzard, S. C. Dudley, and H. Todt Interaction between Fast and Ultra-slow Inactivation in the Voltage-gated Sodium Channel. DOES THE INACTIVATION GATE STABILIZE THE CHANNEL STRUCTURE? J. Biol. Chem., September 27, 2002; 277(40): 37105 - 37115. [Abstract] [Full Text] [PDF]
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V. Yarov-Yarovoy, J. C. McPhee, D. Idsvoog, C. Pate, T. Scheuer, and W. A. Catterall Role of Amino Acid Residues in Transmembrane Segments IS6 and IIS6 of the Na+ Channel alpha Subunit in Voltage-dependent Gating and Drug Block J. Biol. Chem., September 13, 2002; 277(38): 35393 - 35401. [Abstract] [Full Text] [PDF]
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S. M. Sine, H.-L. Wang, and N. Bren Lysine Scanning Mutagenesis Delineates Structural Model of the Nicotinic Receptor Ligand Binding Domain J. Biol. Chem., August 2, 2002; 277(32): 29210 - 29223. [Abstract] [Full Text] [PDF]
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M E O'Leary and M Chahine Cocaine binds to a common site on open and inactivated human heart (Nav1.5) sodium channels J. Physiol., June 15, 2002; 541(3): 701 - 716. [Abstract] [Full Text] [PDF]
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 Y.-F. Xiao, Q. Ke, S.-Y. Wang, K. Auktor, Y. Yang, G. K. Wang, J. P. Morgan, and A. Leaf Single point mutations affect fatty acid block of human myocardial sodium channel alpha subunit Na+ channels PNAS, March 1, 2001; (2001) 61003798. [Abstract] [Full Text]
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S. N. Wright Irreversible Block of Human Heart (hH1) Sodium Channels by the Plant Alkaloid Lappaconitine Mol. Pharmacol., February 1, 2001; 59(2): 183 - 192. [Abstract] [Full Text]
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 C. Nau, M. Seaver, S.-Y. Wang, and G. K. Wang Block of Human Heart hH1 Sodium Channels by Amitriptyline J. Pharmacol. Exp. Ther., March 1, 2000; 292(3): 1015 - 1023. [Abstract] [Full Text]
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A. De Luca, F. Natuzzi, J.-F. Desaphy, G. Loni, G. Lentini, C. Franchini, V. Tortorella, and D. C. Camerino Molecular Determinants of Mexiletine Structure for Potent and Use-Dependent Block of Skeletal Muscle Sodium Channels Mol. Pharmacol., February 1, 2000; 57(2): 268 - 277. [Abstract] [Full Text]
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T. Weiser, Y. Qu, W. A. Catterall, and T. Scheuer Differential Interaction of R-Mexiletine with the Local Anesthetic Receptor Site on Brain and Heart Sodium Channel alpha -Subunits Mol. Pharmacol., December 1, 1999; 56(6): 1238 - 1244. [Abstract] [Full Text]
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C. Nau, S.-Y. Wang, G. R. Strichartz, and G. K. Wang Point Mutations at N434 in D1-S6 of {micro}1 Na+ Channels Modulate Binding Affinity and Stereoselectivity of Local Anesthetic Enantiomers Mol. Pharmacol., August 1, 1999; 56(2): 404 - 413. [Abstract] [Full Text]
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V. Yarov-Yarovoy, J. Brown, E. M. Sharp, J. J. Clare, T. Scheuer, and W. A. Catterall Molecular Determinants of Voltage-dependent Gating and Binding of Pore-blocking Drugs in Transmembrane Segment IIIS6 of the Na+ Channel alpha Subunit J. Biol. Chem., January 5, 2001; 276(1): 20 - 27. [Abstract] [Full Text] [PDF]
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K. Hilber, W. Sandtner, O. Kudlacek, I. W. Glaaser, E. Weisz, J. W. Kyle, R. J. French, H. A. Fozzard, S. C. Dudley, and H. Todt The Selectivity Filter of the Voltage-gated Sodium Channel Is Involved in Channel Activation J. Biol. Chem., July 20, 2001; 276(30): 27831 - 27839. [Abstract] [Full Text] [PDF]
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A. J. Carter, M. Grauert, U. Pschorn, W. D. Bechtel, C. Bartmann-Lindholm, Y. Qu, T. Scheuer, W. A. Catterall, and T. Weiser Potent blockade of sodium channels and protection of brain tissue from ischemia by BIII 890 CL PNAS, April 25, 2000; 97(9): 4944 - 4949. [Abstract] [Full Text] [PDF]
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Y.-F. Xiao, Q. Ke, S.-Y. Wang, K. Auktor, Y. Yang, G. K. Wang, J. P. Morgan, and A. Leaf Single point mutations affect fatty acid block of human myocardial sodium channel alpha subunit Na+ channels PNAS, March 13, 2001; 98(6): 3606 - 3611. [Abstract] [Full Text] [PDF]
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