![[Feedback]](/icons/banner/feedbackACT.gif){kind=icon}
![[For Subscribers]](/icons/banner/subscriberACT.gif){kind=icon}
![[Archive]](/icons/banner/archiveACT.gif){kind=icon}
![[Search]](/icons/banner/searchACT.gif){kind=icon}
![[Contents]](/icons/banner/tocACT.gif){kind=icon}
|
|
|
|
|
||
Departments of Medicine, Section of Molecular and Cellular Cardiology, Johns Hopkins University, Baltimore, Maryland
Received January 24, 2003; accepted June 11, 2003
 |
Abstract |
|---|
 Top Abstract Materials and MethodsResultsDiscussionReferences |
|---|
),
alter the sensitivity of Nav1.4 to lidocaine, independent of
mutation-induced changes in gating. The magnitude of the change in the
IC50 values, the isoform, and LA dependence of the changes in
affinity suggest that the determinants of binding in I-S6 and II-S6 are
subsidiary to those in IV-S6.
subunit that is a large membrane-spanning glycoprotein composed of four
homologous domains (I–IV) and, depending on tissue type, one or more
smaller accessory 
subunits
(Catterall, 2000). Each domain
of the subunit contains six transmembrane segments (S1–S6). A
portion of the cytoplasmic mouth of ion-conducting pore of the Na+
channel is thought to be formed by the carboxyterminal portion of the S6 of
each domain.
Important insights into the structure and function of the outer mouth of
the Na+ channel pore have been revealed by site-directed
mutagenesis (Chiamvimonvat et al.,
1996; Favre et al.,
1996; Perez-Garcia et al.,
1996; Sunami et al.,
1997; Tsushima et al.,
1997; Yamagishi et al.,
1997). However, the constituents of the inner mouth of the pore
are far less certain. Clues to the structure of the inner pore mouth have come
from examination of the structure of a potassium channel from Streptomyces
lividans, KcsA (Doyle et al.,
1998) and from studies of LA block of voltage-dependent
Na+ channels (Gingrich et al.,
1993; Ragsdale et al.,
1994,
1996;
Nau et al., 1999;
Wang and Wang, 1999;
Wang et al., 2000;
Yarov-Yarovoy et al., 2001,
2002).
Local anesthetics are amphiphilic molecules that have been useful molecular
probes of the structure of the Na+ channel
(Gingrich et al., 1993;
Ragsdale et al., 1994,
1996;
Nau et al., 1999;
Wang and Wang, 1999;
Wang et al., 2000;
Yarov-Yarovoy et al., 2001,
2002). Alanine scanning
mutagenesis of IV-S6 of the rat brain (Nav1.2) Na+
channel has identified amino acid residues that form the hydrophobic receptor
subsite for LA binding (Ragsdale et al.,
1994,
1996). F1764A abolished and
I1760A and Y1771A reduced use-dependent etidocaine block. F1764A was the only
mutant that significantly reduced rested state block
(Ragsdale et al., 1994).
F1764A and Y1771A also reduced use-dependent lidocaine block without
significantly affecting the resting channel block
(Ragsdale et al., 1996). The
structural model proposed to rationalize these findings was an -helix
with side chains of the amino acid residues at positions 1760, 1764, and 1771
on the same face of the helix forming the hydrophobic subsite lining the inner
pore of the channel. Lysine mutations at equivalent positions to F1764, N1769,
and Y1771 in the rat skeletal muscle (Nav1.4) Na+
channel altered resting and inactivated channel affinity for cocaine and
benzocaine (Wright et al.,
1998).
A number of studies have implicated amino acid residues in the S6 segments
of other domains in channel gating and LA binding. In I-S6, all substitutions
of the native asparagine at position 434 in Nav1.4 increased the
resting state block by bupivacaine, but only N434K exhibited decreased
inactivated state block (Wang et al.,
1998; Nau et al.,
1999). In I-S6 of Nav1.2, I409A was associated with
decreased inactivated state affinity for etidocaine but not for its tricyclic
congener sipatrigine or the anticonvulsant lamotrigine
(Yarov-Yarovoy et al., 2002).
In II-S6, V787K (Nav1.4) increased the affinity of resting channels
to bupivacaine (Wang et al.,
2001). However, no alanine mutants in II-S6 of Nav1.2
exhibited altered LA blocking affinity
(Yarov-Yarovoy et al., 2002).
L1465A and I1469A in III-S6 of Nav1.2 reduced the affinity of
inactivated channels to lamotrigine; however, these mutations and N1466A
increased the affinity of rested channels to lamotrigine
(Yarov-Yarovoy et al., 2001).
S1276K and L1280K (equivalent to L1465 of Nav1.2) in III-S6 of
Nav1.4 exhibited decreased affinity for bupivacaine and complete
resistance to batrachotoxin (Wang et al.,
2000). It is notable that mutations in all four S6 segments
produced changes in both activation and inactivation gating that can
complicate the interpretation of mutation-induced alterations in local
anesthetic block (Ragsdale et al.,
1994,
1996;
Wright et al., 1998;
Nau et al., 1999;
O'Reilly et al., 2001;
Yarov-Yarovoy et al., 2001,
2002).
We used cysteine scanning mutagenesis to investigate the contribution of
domain I and II S6 segments of the skeletal muscle Nav1.4 channel
to the formation of the inner pore and determine the isoform-specific
contribution of these residues to channel gating and LA block. Our data
suggest that mutation-induced alterations in inactivation gating played an
important role in altering sensitivity to LA block. However, several residues
in I-S6 (Ile424, Ile425, and Gly428) and II-S6 (Ile782 and Val786) altered
lidocaine block of Nav channels independent of any significant
changes in channel gating. A preliminary report of these results has been
published in abstract form (Kondratiev et
al., 2000).
|
Materials and Methods |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
subunit
(Trimmer et al., 1989) cloned
into a vector driving the bicistronic expression of green fluorescent protein
and rNAv1.4 subunit using an internal ribosomal entry site
(IRES) and the rat brain 1 subunit was cloned pCMV-5
(Isom et al., 1992). Mutant
channels were generated polymerase chain reaction using oligonucleotide
primers encoding the mutation of interest using the QuikChange site-directed
tagenesis kit (Stratagene, La Jolla, CA). All mutations were firmed by DNA
sequencing of the region surrounding the mutation and at least two independent
clones of each mutation were studied. HEK293 cells (American Type Culture
Collection, Manassas, were cotransfected with an equimolar ratio of the
Na+ channel subunit and rat brain 1 subunit cDNA using
LipofectAMINE Plus according to the manufacturer's instructions. After
transfection cells were maintained in Dulbecco's modified Eagle's medium
(Invitrogen, Carlsbad, CA) supplemented with 10% fetal bovine serum and
penicillin/streptomycin at 37°C in a 5% CO2-humidified
incubator and used for experiments after 24 h.
Electrophysiology and Electropharmacology. Transfected cells
expressing Na+ channels were identified by epifluorescence
microscopy and voltage-clamped 24 h after transfection using whole-cell
configuration of the patch-clamp technique with an Axopatch 200B patch-clamp
amplifier (Axon Instruments, Inc., Union City, CA) interfaced with a personal
computer. Patch electrodes were pulled from borosilicate glass and had 1- to
2-M
tip resistances. Voltage command protocols were delivered, and data
were acquired using custom-written software. Cell capacitance was calculated
integrating the area under an uncompensated capacity transient elicited by a
10-mV hyperpolarizing test pulse from a holding potential of –70 mV.
Series resistance was then compensated typically 80 to 85%. Currents were
filtered at 5 kHz and digitized with 12-bit resolution. All experiments were
performed 10 min after establishment of the whole-cell configuration to ensure
adequate dialysis of the cell and to minimize the initial rapid time-dependent
shifts in voltage dependence of activation and steady-state inactivation.
Standard voltage protocols were used to generate activation and availability
relationships. A two-pulse protocol was used to assess recovery from
inactivation after short (50-ms) and long (1-s) test depolarizations to
–20 mV; the curves were best fit with two exponentials (with the
exception of N434C).
Mutant channels were screened for changes in local anesthetic sensitivity by comparing the first-pulse block and use-dependent block at a stimulation frequency of 10 Hz (50-ms pulse duration) from a holding potential of –120 mV in the presence of 200 µM lidocaine to the wild-type channel. The magnitude of use-dependent block was the ratio of the steady-state current after 30 pulses in the presence of lidocaine to the current in the absence of drug. Dose-response curves were generated by random application of lidocaine to the bath at concentrations ranging from 1 µM to 20 mM. At least three determinations were made at each lidocaine concentration. All lidocaine block was fully reversible with washout of the drug. The IC50 value was determined by fitting the dose-response curves with a binding isotherm of the form I/I0 = 1/(1 + ([lidocaine]/IC50)), where I and I0 are the peak Na+ currents elicited by a 50-ms voltage step from –120 to –20 mV in the presence and absence of lidocaine, respectively. The reduction in the peak current elicited by the first pulse after a long rest (10 s) at –120 mV was used to determine the tonic block IC50, and the current after 30 pulses at 10 Hz was used to determine the use-dependent IC50.
Solutions. Whole-cell Na+ currents were recorded in an extracellular solution containing 140 mM NaCl, 5 mM KCl, 2 mM CaCl2, 1 mM MgCl2, 10 mM HEPES, pH 7.4. The pipette solution contained 35 mM NaCl, 105 mM CsF, 1 mM MgCl2, 10 mM HEPES, 10 mM EGTA, pH 7.2. Experiments were performed at room temperature (18–22°C). Lidocaine HCl (2-diethylamino-N-[2,6-dimethylphenyl]acetamide; Sigma-Aldrich, St. Louis, MO) was dissolved in the extracellular solution and diluted to the appropriate concentration.
Data Analysis. Pooled data were expressed as mean ± S.E.M. Statistical comparisons were performed using an unpaired Student's t test with p values of <0.05 considered statistically significant. Data were fitted to the appropriate functions by nonlinear least-squares methods (Levenburg-Marquardt algorithm; OriginLab Corp, Northampton, MA).
|
Results |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
1 subunit. The mutations had no significant
effect on the reversal potential. Figure 2,
A and B, shows raw current records and normalized current-voltage
(I-V) relations of mutant Na+ channels that were significantly
shifted compared with wild-type, grouped by domain. I425C, I436C, L785C, and
V787C produced significant depolarizing shifts and V779C a hyperpolarizing
shift of the negative slope region of I-V relations compared with wild type.
The voltage at half-maximal activation (V0.5) of the
conductance-voltage (gNa-V) relations for I425C,
I436C, L785C, and V787C were shifted by +5 to +8 mV compared with wild type
(Fig. 2, C and D;
Table 1). I425C, I432C, and
V787C significantly increased the slopes of gNa-V
curves (Table 1).
|
|
|
Figure 3, A and B, shows the steady-state availability curves for mutant Na+ channels that were significantly shifted compared with wild type. In contrast to the relative paucity of effects on activation, mutations at a number of positions in I-S6 and II-S6 altered steady-state availability. The steady-state availability of three mutants in I-S6 (F426C, F430C, and N434C) and one in II-S6 (V779C) were significantly shifted in the hyperpolarizing direction compared with wild type. Two mutant channels in I-S6 (Y431C and I436C) and three in II-S6 (I782C, V786C, and L790C) exhibited significant depolarizing shifts in availability (Fig. 3, C and D; Table 1). Of the I-S6 mutants that exhibited a shift in the voltages of half-inactivation (V0.5), only N434C had a significantly greater slope of the steady-state availability relation (Table 1). Two mutations in II-S6 (M780C and G783C) significantly increased the slope of availability curves without altering the V0.5, whereas V786C significantly reduced the slope compared with wild type (Table 1).
|
Effect of Mutations on Kinetics of Inactivation. Mutations in I-S6
and II-S6 altered the kinetics of inactivation.
Figure 4, A to D, shows plots
of the fractional recoveries of peak INa as a function of
time at –120 mV after 50-ms (Fig. 4,
A and C) and 1-s (Fig. 4, B and
D) prepulses to –20 mV; the wild-type and mutant channels
that exhibited significantly different kinetics are shown. The recoveries from
inactivation after the 50-ms prepulse (Fig.
4, A and C) were best fit with two exponentials. The dominant fast
time constant was 2.1 ± 0.2 ms comprising 96 ± 11% of the
recovery; the remaining slow component had a time constant of 13.5 ±
2.8 ms for wild-type channels. L427C recovered faster than wild type with a
fast time constant of 1.2 ± 0.1 ms
(Fig. 4A;
Table 2). Two other mutants,
N434C (I-S6) and V779C (II-S6), recovered more slowly than wild type. N434C
exhibited a slowed 
fast (3.3 ± 0.1 ms) of reduced
amplitude (62% of total) compared with wild type. The slow component of
recovery of N434C was enhanced (37% of total) and was more than an order of
magnitude slower (slow = 263 ± 9 ms) than the slow
component of wild type; however, channels still recovered fully in 1 s
(Fig. 4A;
Table 2). V779C recovered with
a dominant fast time constant of 3.2 ± 0.1 ms after 50-ms prepulses
(Fig. 4C;
Table 2).
|
|
To study slow inactivated states, we examined recoveries of the channels
after longer prepulses. A number of mutations slowed the kinetics of recovery
from inactivation after 1-s prepulses with N434C producing the most dramatic
effect (Fig. 4, B and D; Table 2). Wild-type recovery
after long prepulses was best fit with two exponentials: the fast component
(45% of total amplitude) with the time constant of 3.0 ± 0.3 ms and the
slow component (51% of total amplitude) with the time constant of 155 ±
8 ms (Table 2). N434C
(Fig. 4B; Table 2) recovered with a
single-exponential time course that was 3-fold slower than the slow component
of recovery of the wild-type channel ( = 429 ± 78). In addition to
N434C, eight other mutations, six in I-S6 (I424C, L427C, G428C, L432C, I433C,
and I436C) and two in II-S6 (I782C and V786C) slowed the fast time component
of recovery from inactivation without a significant change in the slow
component compared with wild type (Fig. 4,
B and D; Table 2).
Entry into slow inactivated states was assessed using a standard two-pulse
protocol. The time constant for entry into slow inactivated states for wild
type was 1,200 ± 30 ms. A number of mutant Na+ channels in
I-S6 and fewer in II-S6 significantly altered the kinetics of entry into slow
inactivated states (Fig. 4, E and
F; Table 2).
Several mutant channels with slowed recovery kinetics after long prepulses
exhibited faster entry into slow inactivated states (I-S6: I424C, G428C,
L432C, I433C, N434C, and I436C; II-S6: I782C and V786C). Two mutants in I-S6
(S429C and F430C) and one in II-S6 (V781C) delayed the entry into slow
inactivated states (Fig. 4, E and
F; Table 2). The
residual current after a 10-s depolarizing pulse is the component that
resisted entry into slowly recovering inactivated states. The wild-type
channel exhibited a residual current that was 6.1% of the peak; F430C, M780C,
V781C, V787C, L790C, and F791C had significantly greater residual currents and
I424C, F426C, L427C, N434C, and V786C had significantly smaller residual
currents compared with wild type (Table
2).
Lidocaine Block of Na+ Channels. The S6 membrane repeats, particularly in domains III and IV, are important components of local anesthetic binding sites in the Na+ channel. All mutant channels were assessed for their sensitivity to tonic (rested) and use-dependent block by lidocaine. Lidocaine was continuously added to the bath for 10 min before beginning of recordings. With infrequent stimulation (0.1 Hz) lidocaine (200 µM) reduced maximum Na+ conductance of the wild-type channels by 19 ± 2% (Fig. 5A; Table 3). G428C (I-S6) exhibited significantly reduced block of gNa,max by lidocaine compared with wild type, whereas N434C, I436C, L785C, and V787C displayed enhanced rested state block (Fig. 5A).
|
|
Local anesthetic block is conformation-sensitive with the drug
preferentially binding to the open and inactivated states
(Hille, 1977;
Hondghem and Katzung, 1977;
Balser et al.,
1996,Balser et al.,
1996). At 10-Hz stimulation frequency (50-ms pulse durations and
50-ms recovery periods) from a holding potential of –120 mV, 200 µM
lidocaine reduced the peak wild-type current by 68 ± 2%
(Fig. 5A). F430C, I782C, and
V786C exhibited significantly attenuated block at rapid rates of stimulation
compared with wild type (Fig.
5A).
Dose-response curves were determined for mutant channels that exhibited significantly altered basic biophysical properties or sensitivity to 200 µM lidocaine compared with wild type. The IC50 for tonic block of G428C by lidocaine was 2-fold greater than the IC50 for wild type, whereas the IC50 values for N434C, I436C, and L785C mutant channels were 40 to 60% lower (Fig. 5B; Table 3). The IC50 values for use-dependent block of I424C, I425C, F430C, I782C, and V786C were more than 2-fold greater than the IC50 value for wild type (Fig. 5B; Table 3).
Effect of Lidocaine on Kinetics and Voltage Dependence of Gating.
Apparent differences in lidocaine affinity may result from changes in the
voltage dependence or kinetics of channel gating
(Nuss et al., 2000). A number
of cysteine substitutions in I-S6 and II-S6 produced changes in the voltage
dependence and kinetics of gating (Figs.
2,
3,
4; Tables
1 and
2). Lidocaine shifted all of
the steady-state availability curves at least 10 mV in the hyperpolarizing
direction compared with drug-free controls
(Fig. 6;
Table 4) but had more modest
and inconsistent effects on the activation curves
(Table 4). The
V0.5 of activation and steady-state inactivation for both
I-S6 and II-S6 mutant channels were not significantly different from wild-type
in the presence of lidocaine with few exceptions. I425C, I436C, L785C, and
N789C had significantly depolarized activation V0.5 values
(Table 4). F426C and N434C had
significantly hyperpolarized availability V0.5s in the
presence of lidocaine compared with wild type, whereas Y431C, I782C, and V786C
significantly had depolarized availability V0.5s in the
presence of lidocaine compared with wild type
(Table 4). In general,
lidocaine produced shifts of similar magnitude in the voltage dependence of
the steady-state availability relations in mutant and wild-type channels. The
only exception was N434C that exhibited an exaggerated hyperpolarizing shift
in the availability curve (Fig.
6A).
|
|
Lidocaine slowed the recovery from inactivation and hastened the entry into the inactivated states increasing the kinetic complexity of both processes. We used the fractional current at 50 ms (F50) as a model-independent index of the rate of recovery from (FR50) and entry into (FI50) inactivated states in the absence and presence of 200 µM lidocaine. In the presence of drug, F430C exhibited hastened recovery from inactivation and slowed the entry into inactivated states (Fig. 7, A and C). Similar to F430C, two mutants in II-S6, I782C and V786C, exhibited hastened recovery after 50-ms depolarizations (V786C) and slowed entry into inactivated states (I782C and V786C; Fig. 7, A and C). Despite dramatic enhancement of the rate of entry and slowed recovery from inactivation in the absence of drug (Fig. 4; Table 2), the entry rate (Fig. 7C) and recovery after short pulses (Fig. 7A) for N434C were not significantly different from wild type in the presence of lidocaine. However, recoveries after 1-s prepulses were significantly slower for N434C and L435C compared with wild type (Fig. 7B).
|
|
Discussion |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
,
2001), replacement of residues
in I-S6 and II-S6 segments with cysteine was well tolerated; all mutant
channels were functional when the subunits were coexpressed with the
1 subunits in HEK293 cells. Our data reveal that a distinct pattern of
mutant channels that display significant changes in the voltage dependence and
kinetics of gating compared with wild-type Nav1.4.
Rotational movement of the M2 (analogous to S6) segment accompanies opening
of the KcsA channel (Perozo et al.,
1999). Similarly, activation of voltage-dependent channels has
been associated with movements of the S6 segments; indeed these regions likely
underlie the activation gate (Liu et al.,
1997; Holmgren et al.,
1998; del Camino et al.,
2000; Li-Smerin et al.,
2000). Mutations of Nav1.4 in I-S6 and II-S6 that
altered activation gating (I425C, I436C, L785C, and V787C) shift the curves in
the depolarizing direction as if substituting these hydrophobic residues with
cysteine eliminated or generated new intramolecular contacts that stabilized
the closed state (Fig. 2). A
similar depolarizing shift and reduction in the slope of V787C has been
described (O'Reilly et al.,
2001). In contrast to N434C, the alanine substitution at position
434 in Nav1.4 produced a depolarizing shift in the activation curve
(Wang and Wang, 1997;
Nau et al., 1999). This
pattern of alterations in the voltage dependence of activation is distinct
from that of alanine scanning mutagenesis of analogous residues of I-S6 and
II-S6 in Nav1.2. In Nav1.2, only the alanine
substitutions at positions 418 and 975 (analogous to Asn434 and Leu788 in
Nav1.4) exhibited significant depolarizing shifts in the activation
curve (Yarov-Yarovoy et al.,
2002).
The discordance of the effects of some of the mutations on activation and
inactivation gating suggests that the S6 segments undergo conformational
changes during inactivation gating that are not simply linked to activation
even though opening and inactivation are coupled processes in Na+
channels (Armstrong and Bezanilla,
1977; Chahine et al.,
1994; Yang and Horn,
1995; Yang et al.,
1996). I436C on the cytoplasmic side of I-S6 exhibited activation
and steady-state availability curves that were shifted in the depolarizing
direction (Figs. 2 and
3;
Table 1), consistent with
resistance to channel opening and subsequent inactivation. I425C on the
extracellular side of I-S6 had a similar shift in the activation curve and a
smaller shift in the availability curve in the same direction. V779C on the
extracellular side of II-S6 exhibited hyperpolarizing shifts in the activation
and availability curves suggesting that this mutation destabilized the closed
state and promoted closed state inactivation (Figs.
2 and
3;
Table 1). Mutations separated
by four residues in I-S6 (F426C, F430C, and N434C) and II-S6 (I782C, V786C,
and L790C) exhibited significant hyperpolarizing and depolarizing shifts in
the voltage dependence of steady-state availability, respectively
(Fig. 3). This pattern suggests
that a face of the -helices in I-S6 and II-S6 stabilizes and
destabilizes inactivation, respectively. This would be consistent with
rotation and/or translation of the S6 helices during inactivation. Our results
differ from that of alanine mutants in the analogous region of
Nav1.2 expressed in Xenopus laevis oocytes where small
shifts of the half-inactivation voltages, mostly in the depolarizing direction
were observed (Yarov-Yarovoy et al.,
2002). The substituting amino acid has profound effects on the
magnitude and direction of the gating shift observed in S6 mutant channels
(Nau et al., 1999), consistent
with the notion that a number of interactions are altered during movement of
the S6 during inactivation gating.
A number of kinetically distinct inactivated states have been described in
Na+ channels (Bezanilla and
Armstrong, 1977; Balser et al.,
1996,Balser et al.,
1996; Featherstone et al.,
1996; Hayward et al.,
1997; Benitah et al.,
1999). Mutations in I-S6 and II-S6 significantly altered the rate
at which the channels enter or recover from inactivated states
(Fig. 4). Longer
depolarizations favored the recruitment of slower forms of inactivation. The
precise mechanism of slow inactivation in Na+ channels is not known
but residues in I-S6 and II-S6 altered the kinetics of these processes. The
effects were particularly prominent in I-S6 where six of the 13 mutants
delayed recovery from inactivation after long pulses and hastened entry into
inactivated states (Figs. 4 and
7) seemingly favoring occupancy
of slowly recovering inactivated states. Arguably, the most dramatic
inactivation gating changes were observed with mutation of a highly conserved
asparagine in I-S6 (N434C). Conserved asparagines are present at
sequence-aligned positions in the S6 segments of all four domains: Asn434 (I),
Asn789 (II), N1281 (III), and N1584 (IV). N434C uniquely modulated the voltage
dependence and kinetics of channel gating stabilizing inactivated states,
whereas N789C produced no significant changes in the voltage dependence or
kinetics of gating. Our data are consistent with previous reports that have
highlighted the importance of Asn434 in channel gating (Wang and Wang,
1997,
1998;
Nau et al., 1999).
Substitutions at position 434 in Nav1.4 with aromatic amino acids
and cysteine produced large hyperpolarizing shifts in the availability curves,
whereas substitution with polar and charged residues tended to produce small
depolarizing shifts (Nau et al.,
1999). Hydrophobic interactions involving the residue at position
434 seem to stabilize inactivation. Replacement of the asparagines in III-S6
and IV-S6 of Nav1.2 also produced significant effects on the
voltage dependence and kinetics of gating that resulted in altered sensitivity
to LAs (Ragsdale et al., 1994;
Wright et al., 1998;
Yarov-Yarovoy et al., 2001).
Alanine substitutions in III-S6 and IV-S6 caused significant hyperpolarizing
shifts of availability curves and increased rested state sensitivity to
etidocaine (Ragsdale et al.,
1994) and lamotrigine
(Yarov-Yarovoy et al., 2001),
whereas a large depolarizing shift of the availability curve of N1584K was
associated with less rested and inactivated state LA block
(Wright et al., 1998).
Lidocaine Block of Cysteine Mutants in I-S6 and II-S6. The
cytoplasmic portion of S6 is presumed to line in the inner mouth of the
channel if the structure of voltage-gated ion channels is analogous to that of
KcsA (Doyle et al., 1998;
Lipkind and Fozzard, 2000).
Mutations of S6 residues in all four domains alter LA block of the
Na+ channel (Ragsdale et al.,
1994; Wright et al.,
1998; Nau et al.,
1999; Wang et al.,
2001; Yarov-Yarovoy et al.,
2001,
2002). Prominent
destabilization of LA block occurs with mutations in IV-S6 (Ragsdale et al.,
1994,
1996) and to a lesser extent
III-S6 (Yarov-Yarovoy et al.,
2001). The role that I-S6 and II-S6 play in LA binding is less
certain. However, recent alanine scanning mutagenesis of I-S6 and II-S6 in the
Nav1.2 channel reveals a single mutation (I409A) that selectively
reduced the affinity for etidocaine
(Yarov-Yarovoy et al., 2002).
Other studies in the Nav1.4 channel background have demonstrated
the importance of residues in I-S6 in LA block and have suggested that the
quaternary ammonium interacts with Asn434
(Wang et al., 1998;
Nau et al., 1999).
Our data implicate a number of other residues in I-S6 and II-S6 of
Nav1.4 in LA block. The interpretation of mutation-induced
alterations in LA block is often complicated by concomitant changes in channel
gating. In fact, given the extent of changes in channel gating demonstrated by
I-S6 and II-S6 cysteine mutants in the absence of drug, it is notable that
larger changes in the sensitivity to block by lidocaine were not observed. It
is likely that mutations in this region of the channel concomitantly alter
both gating and drug binding (Wang et al.,
1998; Nau et al.,
1999; Yarov-Yarovoy et al.,
2002). The effects of the mutations that significantly altered
lidocaine sensitivity were modest with the IC50 values for tonic
and use-dependent block of mutants differing by less than a factor of 3
(Fig. 5B; Table 3;
Wang et al., 1998). Several
mutant channels reduced the sensitivity to use-dependent lidocaine block and
none significantly enhanced sensitivity
(Table 3). The reduced
use-dependent block was associated with slower entry and faster recovery from
inactivated states in the presence of lidocaine (F430C). However, other
mutants with increased IC50 values for use-dependent block (I424C,
I425C, I782C, and V786C) did not exhibit gating changes that could explain the
reduced sensitivity to lidocaine, suggesting that LA binding is altered in
these mutants. G428C was uniquely resistant to first-pulse block
(Fig. 5), yet underwent
inactivation more rapidly and recovery more slowly than the wild-type channel
(Fig. 4, B and E). Mutants with
enhanced first-pulse block can be divided into those with gating changes that
served to explain the enhanced sensitivity (N434C and I436C) and those that
exhibited no significant differences in gating (L785C and V787C) accounting
for the increased rested state affinity for lidocaine, although enhanced drug
access in the rested state and other mutation-induced structural changes
cannot be excluded by our data (Figs.
5,
6,
7;
Table 4).
Orthodoxy in LA block of Na+ channels predicts that
stabilization of inactivation should enhance block, particularly use-dependent
block. Thus, the enhanced lidocaine block of mutant channels such as N434C is
likely the result of mutation-induced changes in inactivation gating. However,
other mutants that stabilized steady-state inactivation (F426C, F430C, and
V779C) failed to enhance or even reduced rested state or use-dependent
sensitivity to lidocaine (Figs.
3,
5, and
7). Lidocaine block may also
rely on the occupancy of slowly recovering inactivated states
(Balser et al.,
1996,Balser et al.,
1996). F430C resisted the entry into slow inactivated states,
recovered more rapidly from inactivation
(Fig. 4) in the presence of
lidocaine, and exhibited reduced use-dependent block
(Fig. 5). Similarly, enhanced
rested block seen for N434C likely resulted from enhanced slow inactivation.
This is similar to the previously reported effects of bupivacaine on N434C
(Nau et al., 1999).
Counterexamples include I782C and V786C, which exhibited reduced sensitivity
to use-dependent block despite stabilization of slowly recovering inactivated
states manifested by hastened recovery from and entry into these states
(Fig. 4).
Our data are consistent with movement of the S6 -helices during
activation and inactivation gating. Changes in the position of the side chains
of S6 residues during gating may alter channel-drug interactions and thereby
modify sensitivity to block by LAs. Indeed, fundamentally different
physicochemical mechanisms may characterize the molecular interactions between
LAs and varying states of the Na+ channel
(Li et al., 1999).
Alternatively, mutations in I-S6 and II-S6 may produce allosteric changes in
the drug receptor conformation and channel gating machinery. In the context of
previously published site-specific mutagenesis data (Ragsdale et al.,
1994,
1996;
Nau et al., 1999;
Wang et al., 1998;
Yarov-Yarovoy et al., 2001,
2002) and our own, we suggest
the topological relationship of the S6 segments of the Na+ channel
shown in Fig. 8. This model is
largely in agreement with that of Yarov-Yarovoy et al.
(2002) but differs from
previously published models. In particular, the changes in LA affinity of
N434C does not mandate a position of this side chain in the pore of the
channel. This model suggests that binding is highly asymmetric with I-S6 and
II-S6 being quantitatively less important than IV-S6 and III-S6, where potent
-stacking and -quaternary ammonium interactions are likely to
occur.
|
The seemingly contradictory data regarding the role of individual residues
in S6 can be reconciled by consideration of the differing experimental
approaches (Ragsdale et al.,
1994,
1996;
Wang et al., 1998;
Wright et al., 1998;
Nau et al., 1999;
Yarov-Yarovoy et al., 2001,
2002). The detailed structure
of the binding sites may vary in different Na+ channel isoforms.
Importantly, utilization of mutagenic strategies to determine which residues
influence LA binding depends to a significant degree on the substituting amino
acid and the structure of the pharmacophore.
|
Acknowledgements |
|---|
|
Footnotes |
|---|
ABBREVIATIONS: HEK, human embryonic kidney; I-V, current-voltage.
Address correspondence to: Dr. Gordon F. Tomaselli, 720 N. Rutland Ave., Ross 844, Johns Hopkins University, Baltimore, MD 21205. E-mail: gtomasel{at}jhmi.edu
|
References |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
Balser JR, Nuss HB, Orias DW, Johns DC, Marban E, Tomaselli GF, and Lawrence JH (1996) Local anesthetics as effectors of allosteric gating. Lidocaine effects on inactivation-deficient rat skeletal muscle Na channels. J Clin Investig 98: 2874–2886.[Medline]
Balser JR, Nuss HB, Romashko DN, Marban E, and Tomaselli GF
(1996) Functional consequences of lidocaine binding to
slow-inactivated sodium channels. J Gen Physiol
107:
643–658.
Benitah JP, Chen Z, Balser JR, Tomaselli GF, and Marban E
(1999) Molecular dynamics of the sodium channel pore vary with
gating: interactions between P-segment motions and inactivation. J
Neurosci 19:
1577–1585.
Bezanilla F and Armstrong CM (1977) Inactivation of
the sodium channel. I. Sodium current experiments. J Gen
Physiol 70:
549–566.
Catterall WA (2000) From ionic currents to molecular mechanisms: the structure and function of voltage-gated sodium channels. Neuron 26: 13–25.[CrossRef][Medline]
Chahine M, George AL Jr, Zhou M, Ji S, Sun W, Barchi RL, and Horn R (1994) Sodium channel mutations in paramyotonia congenita uncouple inactivation from activation. Neuron 12: 281–294.[CrossRef][Medline]
Chiamvimonvat N, Perez-Garcia MT, Ranjan R, Marban E, and Tomaselli GF (1996) Depth asymmetries of the pore-lining segments of the Na+ channel revealed by cysteine mutagenesis. Neuron 16: 1037–1047.[CrossRef][Medline]
del Camino D, Holmgren M, Liu Y, and Yellen G (2000) Blocker protection in the pore of a voltage-gated K+ channel and its structural implications. Nature (Lond) 403: 321–325.[CrossRef][Medline]
Doyle DA, Morais Cabral J, Pfuetzner RA, Kuo A, Gulbis JM, Cohen
SL, Chait BT, and MacKinnon R (1998) The structure of the
potassium channel: molecular basis of K+ conduction and
selectivity. Science (Wash DC)
280:
69–77.
Favre I, Moczydlowski E, and Schild L (1996) On the structural basis for ionic selectivity among Na+, K+, and Ca2+ in the voltage-gated sodium channel. Biophys J 71: 2916–2918.[Medline]
Featherstone DE, Richmond JE, and Ruben PC (1996) Interaction between fast and slow inactivation in Skm1 sodium channels. Biophys J 71: 3098–3109.[Medline]
Gingrich KJ, Beardsley D, and Yue DT (1993) Ultra-deep
blockade of Na+ channels by a quaternary ammonium ion: catalysis by
a transition-intermediate state? J Physiol (Lond)
471:
319–341.
Hayward LJ, Brown RH Jr, and Cannon SC (1997) Slow inactivation differs among mutant Na channels associated with myotonia and periodic paralysis. Biophys J 72: 1204–1219.[Medline]
Hille B (1977) Local anesthetics: hydrophilic and
hydrophobic pathways for the drug-receptor reaction. J Gen
Physiol 69:
497–515.
Holmgren M, Shin KS, and Yellen G (1998) The activation gate of a voltage-gated K+ channel can be trapped in the open state by an intersubunit metal bridge. Neuron 21: 617–621.[CrossRef][Medline]
Hondghem LM and Katzung BG (1977) A unifying molecular model for the interaction of antiarrhythmic drugs with cardiac sodium channels: application to quinidine and lidocaine. Proc West Pharmacol Soc 20: 253–256.[Medline]
Isom LL, De Jongh KS, Patton DE, Reber BF, Offord J, Charbonneau H,
Walsh K, Goldin AL, and Catterall WA (1992) Primary structure and
functional expression of the beta 1 subunit of the rat brain sodium channel.
Science (Wash DC) 256:
839–842.
Kondratiev A, Mendez A, and Tomaselli GF (2000) Amino acids in the S6 segments of domains I and II of the Na channel modulate local anesthetic drug block (Abstract). Circulation 102: 49788.
Lipkind GM and Fozzard HA (2000) KcsA crystal structure as framework for a molecular model of the Na+ channel pore. Biochemistry 39: 8161–8170.[CrossRef][Medline]
Li HL, Galue A, Meadows L, and Ragsdale DS (1999) A
molecular basis for the different local anesthetic affinities of resting
versus open and inactivated states of the sodium channel. Mol
Pharmacol 55:
134–141.
Li-Smerin Y, Hackos DH, and Swartz KJ (2000) A localized interaction surface for voltage-sensing domains on the pore domain of a K+ channel. Neuron 25: 411–423.[CrossRef][Medline]
Liu Y, Holmgren M, Jurman ME, and Yellen G (1997) Gated access to the pore of a voltage-dependent K+ channel. Neuron 19: 175–184.[CrossRef][Medline]
Nau C, Wang SY, Strichartz GR, and Wang GK (1999)
Point mutations at N434 in D1–S6 of mu1 Na+ channels modulate
binding affinity and stereoselectivity of local anesthetic enantiomers.
Mol Pharmacol 56:
404–413.
Nuss HB, Kambouris NG, Marban E, Tomaselli GF, and Balser JR (2000) Isoform-specific lidocaine block of sodium channels explained by differences in gating. Biophys J 78: 200–210.[Medline]
O'Reilly JP, Wang SY, and Wang GK (2001) Residue-specific effects on slow inactivation at V787 in D2–S6 of Na(v)1.4 sodium channels. Biophys J 81: 2100–2111.[Medline]
Perez-Garcia MT, Chiamvimonvat N, Marban E, and Tomaselli GF
(1996) Structure of the sodium channel pore revealed by serial
cysteine mutagenesis. Proc Natl Acad Sci USA
93:
300–304.
Perozo E, Cortes DM, and Cuello LG (1999) Structural
rearrangements underlying K+-channel activation gating.
Science (Wash DC) 285:
73–78.
Ragsdale DS, McPhee JC, Scheuer T, and Catterall WA
(1994) Molecular determinants of state-dependent block of
Na+ channels by local anesthetics. Science (Wash
DC) 265:
1724–1728.
Ragsdale DS, McPhee JC, Scheuer T, and Catterall WA
(1996) Common molecular determinants of local anesthetic,
antiarrhythmic and anticonvulsant block of voltage-gated Na+
channels. Proc Natl Acad Sci USA
93:
9270–9275.
Sunami A, Dudley SC, and Fozzard HA (1997) Sodium
channel selectivity filter regulates antiarrhythmic drug binding.
Proc Natl Acad Sci USA
94:
14126–14131.
Trimmer JS, Cooperman SS, Tomiko SA, Zhou JY, Crean SM, Boyle MB, Kallen RG, Sheng ZH, Barchi RL, Sigworth FJ, et al. (1989) Primary structure and functional expression of a mammalian skeletal muscle sodium channel. Neuron 3: 33–49.[CrossRef][Medline]
Tsushima RG, Li RA, and Backx PH (1997) P-loop
flexibility in Na+ channel pores revealed by single- and
double-cysteine replacements. J Gen Physiol
110:
59–72.
Wang GK, Quan C, and Wang SY (1998) Local anesthetic
block of batrachotoxin-resistant muscle Na+ channels.
Mol Pharmacol 54:
389–396.
Wang SY, Barile M, and Wang GK (2001) Disparate role
of Na+ channel D2–S6 residues in batrachotoxin and local
anesthetic action. Mol Pharmacol
59:
1100–1107.
Wang SY, Nau C, and Wang GK (2000) Residues in Na+ channel D3–S6 segment modulate both batrachotoxin and local anesthetic affinities. Biophys J 79: 1379–1387.[Medline]
Wang SY and Wang GK (1997) A mutation in segment I-S6 alters slow inactivation of sodium channels. Biophys J 72: 1633–1640.[Medline]
Wang SY and Wang GK (1998) Point mutations in segment
I-S6 render voltage-gated Na+ channels resistant to batrachotoxin.
Proc Natl Acad Sci USA
95:
2653–2658.
Wang SY and Wang GK (1999) Batrachotoxin-resistant Na+ channels derived from point mutations in transmembrane segment D4–S6. Biophys J 76: 3141–3149.[Medline]
Wright SN, Wang SY, and Wang GK (1998) Lysine point
mutations in Na+ channel D4–S6 reduce inactivated channel
block by local anesthetics. Mol Pharmacol
54:
733–739.
Yamagishi T, Janecki M, Marban E, and Tomaselli GF (1997) Topology of the P segments in the sodium channel pore revealed by cysteine mutagenesis. Biophys J 73: 195–204.[Medline]
Yang N, George AL Jr, and Horn R (1996) Molecular basis of charge movement in voltage-gated sodium channels. Neuron 16: 113–122.[CrossRef][Medline]
Yang N and Horn R (1995) Evidence for voltage-dependent S4 movement in sodium channels. Neuron 15: 213–218.[CrossRef][Medline]
Yarov-Yarovoy V, Brown J, Sharp EM, Clare JJ, Scheuer T, and
Catterall WA (2001) Molecular determinants of voltage-dependent
gating and binding of pore-blocking drugs in transmembrane segment IIIS6 of
the Na+ channel subunit. J Biol
Chem 276:
20–27.
Yarov-Yarovoy V, McPhee JC, Idsvoog D, Pate C, Brown J, Scheuer T,
and Catterall WA (2002) Role of amino acid residues in
transmembrane segments IS6 and IIS6 of the Na+ channel
subunit in voltage-dependent gating and drug block. J Biol
Chem 277:
35393–35401.
This article has been cited by other articles:
|
|
 |
|
  L. E. Browne, F. E. Blaney, S. P. Yusaf, J. J. Clare, and D. Wray Structural Determinants of Drugs Acting on the Nav1.8 Channel J. Biol. Chem., April 17, 2009; 284(16): 10523 - 10536. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. A. Ahern, A. L. Eastwood, D. A. Dougherty, and R. Horn Electrostatic Contributions of Aromatic Residues in the Local Anesthetic Receptor of Voltage-Gated Sodium Channels Circ. Res., January 4, 2008; 102(1): 86 - 94. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. H. Chancey, P. E. Shockett, and J. P. O'Reilly Relative resistance to slow inactivation of human cardiac Na+ channel hNav1.5 is reversed by lysine or glutamine substitution at V930 in D2-S6 Am J Physiol Cell Physiol, December 1, 2007; 293(6): C1895 - C1905. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. M. McNulty, J. W. Kyle, G. M. Lipkind, and D. A. Hanck An Inner Pore Residue (Asn406) in the Nav1.5 Channel Controls Slow Inactivation and Enhances Mibefradil Block to T-Type Ca2+ Channel Levels Mol. Pharmacol., November 1, 2006; 70(5): 1514 - 1523. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 W. Ulbricht Sodium Channel Inactivation: Molecular Determinants and Modulation Physiol Rev, October 1, 2005; 85(4): 1271 - 1301. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Y. Tsang, R. G. Tsushima, G. F. Tomaselli, R. A. Li, and P. H. Backx A Multifunctional Aromatic Residue in the External Pore Vestibule of Na+ Channels Contributes to the Local Anesthetic Receptor Mol. Pharmacol., February 1, 2005; 67(2): 424 - 434. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
 |
 |
 |
|
 |
 |
 |
