§
From the * Center for Molecular Recognition, Department of Neurology, § Department of Physiology and Cellular Biophysics, and
Department of Biochemistry and Molecular Biophysics, Columbia University, New York 10032
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ABSTRACT |
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The triethylammonium QX-314 and the trimethylammonium QX-222 are lidocaine derivatives that
act as open-channel blockers of the acetylcholine (ACh) receptor. When bound, these blockers should occlude
some of the residues lining the channel. Eight residues in the second membrane-spanning segment (M2) of the
mouse-muscle subunit were mutated one at a time to cysteine and expressed together with wild-type
,
, and
subunits in Xenopus oocytes. The rate constant for the reaction of each substituted cysteine with 2-aminoethyl
methanethiosulfonate (MTSEA) was determined from the time course of the irreversible effect of MTSEA on the ACh-induced current. The reactions were carried out in the presence and absence of ACh and in the presence
and absence of QX-314 and QX-222. These blockers had no effect on the reactions in the absence of ACh. In the
presence of ACh, both blockers retarded the reaction of extracellularly applied MTSEA with cysteine substituted
for residues from
Val255, one third of the distance in from the extracellular end of M2, to
Glu241, flanking the
intracellular end of M2, but not with cysteine substituted for
Leu258 or
Glu262, at the extracellular end of M2.
The reactions of MTSEA with cysteines substituted for
Leu258 and
Glu262 were considerably faster in the presence of ACh than in its absence. That QX-314 and QX-222 did not protect
L258C and
E262C against reaction
with MTSEA in the presence of ACh implies that protection of the other residues was due to occlusion of the
channel and not to the promotion of a less reactive state from a remote site. Given the 12-Å overall length of the blockers and the
-helical conformation of M2 in the open state, the binding site for both blockers extends from
Val255 down to
Ser248.
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INTRODUCTION |
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There is considerable evidence that certain noncompetitive inhibitors (NCIs)1 of the acetylcholine receptor
act as open-channel blockers, binding within the open
channel and occluding it (Adams, 1976; Ruff, 1977
; Ascher et al., 1978
; Neher and Steinbach, 1978
; Colquhoun and Sheridan, 1981
; Farley et al., 1981
). NCIs,
binding either within the channel or at lower affinity
sites (Krodel et al., 1979
; Heidmann et al., 1983
; Herz
et al., 1987
; Lurtz et al., 1997
), can also interact with
closed states of the receptor (Adams, 1976
; Farley et al.,
1981
; Neher, 1983
) and promote desensitization (Magleby and Pallotta, 1981
; Karpen et al., 1982
; Maleque
et al., 1982
; Sine and Taylor, 1982
; Neher, 1983
; Oswald et al., 1983
; Boyd and Cohen, 1984
; Clapham and
Neher, 1984
; Gage and Wachtel, 1984
). Based on the
likelihood that NCIs bind within the channel, reactive NCI derivatives were used to identify channel-lining
residues. Chlorpromazine (Revah et al., 1990
), triphenylmethylphosphonium (Hucho et al., 1986
), and meproadifen mustard (Pedersen et al., 1992
) all labeled
residues in or flanking the predicted second membrane-spanning segment (M2) (Fig. 1) of one or more
Torpedo species acetylcholine (ACh)-receptor subunits
(Fig. 2). In particular, the labeling of
S248 and the
aligned residues in
,
, and
by chlorpromazine and
triphenylmethylphosphonium (Fig. 2) provided evidence that the channel was on the axis of the pentameric complex (
2
) surrounded by the five M2 segments.
|
|
Other crucial evidence for the topology of M2 and
for its central role in the channel was provided by the
effects on conductance and selectivity of mutating
charged residues flanking M2 in each of the subunits
(Imoto et al., 1988). In addition, the effects of mutations of the M2 residues
S248 and
S252 and of the
aligned residues in the other subunits (Fig. 2) on the
kinetics of QX-222 block (Fig. 3) provided further evidence that open-channel blockers bind in the channel
in the vicinity of these M2 residues (Charnet et al.,
1990
; Leonard et al., 1988
). Furthermore, the mutation L247T in the homopentameric neuronal ACh receptor
formed by the
7 subunit (Anand et al., 1991
; Cooper
et al., 1991
) resulted in the loss of sensitivity to block by
QX-222 (Revah et al., 1991
). (
7)L247 aligns with
mouse-muscle
L251 (Fig. 2).
|
Not all evidence, however, points to the region between S248 and
S252 as the sole binding site in the
channel. Quinacrine azide photolabeled residues at the
extracellular end of
M1, specifically in the open state
of the channel (Karlin, 1989
; DiPaola et al., 1990
), and
mutations at the extracellular end of
M1 affected quinacrine but not chlorpromazine binding (Tamamizu et al., 1995
). Nevertheless, chlorpromazine and a
number of other NCIs retarded the labeling by quinacrine azide (DiPaola et al., 1990
) and competed with
the binding of quinacrine (Lurtz et al., 1997
). In addition, meproadifen mustard labeled
E262 at the extracellular end of M2 (Pedersen et al., 1992
). Different
NCIs may bind to nonidentical, possibly overlapping
sites in the channel.
We report here on another approach to the localization of the sites of action of channel-blocking NCIs,
using them in conjunction with the substituted-cysteine-accessibility method (SCAM). SCAM was applied previously to the and
subunits, identifying residues exposed in the channel along the entire length of M2
(Fig. 2), in the M1-M2 loop, and in the extracellular
third of M1 (Akabas et al., 1992
, 1994
; Akabas and Karlin, 1995
; Zhang and Karlin, 1997
, 1998
; Wilson and Karlin, 1998
). These residues, when substituted by cysteine,
reacted with small, positively charged sulfhydryl-specific methanethiosulfonate reagents such as 2-aminoethyl
methanethiosulfonate (MTSEA). The rate constants for
the reactions with the different residues depended on
the state of the receptor (Pascual and Karlin, 1998
) and
on the side of application of the reagent (Wilson and Karlin, 1998
). In this paper, we test to what extent the
channel blockers QX-222 and QX-314 (Fig. 3; Neher
and Steinbach, 1978
; Horn et al., 1980
; Neher, 1983
;
Charnet et al., 1990
) protect Cys-substituted channel-lining residues in
M2 from reaction with extracellularly applied MTSEA. Previously, Cys-substituted residues in the M2 segment of the GABAA receptor
1 subunit
were protected by picrotoxin (Xu et al., 1995
), Cys-substituted residues in the Na channel were protected by
tetrodotoxin (Kirsch et al., 1994
), Cys-substituted residues in Kv2.1 potassium channel were protected by tetraethylammonium (Pascual et al., 1995
), and Cys-substituted residues in Shaker potassium channel were protected by agitoxin (Gross and MacKinnon, 1996
). We
find that QX-222 and QX-314, in the presence of ACh,
retard the reactions of extracellularly added MTSEA
with Cys-substituted residues from
V255C (Fig. 2) down to the cytoplasmic end of
M2.
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METHODS |
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Materials
The quaternary lidocaine derivative 2-(triethylammonio)-N-(2,6-dimethylphenyl)acetamide bromide (QX-314) was from Alomone Laboratories (Jerusalem, Israel) and from Astra (Westborough, MA), and 2-(trimethylammonio)-N-(2,6-dimethylphenyl)acetamide chloride (QX-222) was from Astra. MTSEA was synthesized as previously described (Stauffer and Karlin, 1994) and purchased
from Toronto Research Chemicals (Toronto, Ontario, Canada).
2-Hydroxyethyl methanethiosulfonate (MTSEH) was synthesized
as described previously (Pascual and Karlin, 1998
).
Mutagenesis and Expression
All mutations were introduced in the M2 segment of the mouse
muscle subunit, and capped, runoff cRNA transcripts were obtained for the
-subunit mutants and for wild-type
,
,
, and
subunits after linearization of the plasmid cDNA, using the
mMessage mMachine kit (Ambion Inc., Austin, TX). cRNAs at a
concentration of 1 mg/ml in water were stored at
80°C. They
were diluted and mixed for injection at a ratio of 2
:1
:1
:1
.
Stage V and VI Xenopus laevis oocytes were collected and defolliculated in collagenase following standard procedures (Akabas et
al., 1992
). Oocytes were injected with 60 nl of cRNA diluted to
1-100 ng/µl, depending on desired current expression levels.
Cells were kept in culture and used for recording on days 1-10.
Two-Electrode Voltage Clamp Recording
Currents were recorded under two-electrode voltage clamp. The
oocyte bath solution contained (mM): 115 NaCl, 2.5 KCl, 1.8 MgCl2, 10 HEPES, pH 7.2, except where indicated otherwise. Solutions flowed at 7 ml/min first through a stainless steel coil immersed in a thermostat at 18.0°C, and then past the oocyte, which
was held in a rectangular chamber with a cross-section normal to
the direction of solution flow of 4 mm2. An agar bridge connected a Ag:AgCl reference electrode to the bath and was placed
as close as possible to the oocyte. The bath was clamped at
ground potential. We used beveled agarose-cushion (Schreibmayer et al., 1994) glass micropipettes filled with 3 M KCl, of resistance ~0.2-0.5 M
, for both current-passing and voltage-
recording electrodes. A few uninjected oocytes from each batch
were tested for the presence of endogenous ACh-induced currents, which were never found. The function of wild-type and
mutant receptors was assayed as the ACh-induced current elicited
by the application of brief (3-25 s) pulses of ACh, at a concentration 10× the EC50, as determined for each mutant, and at a holding potential of
50 mV, except where indicated otherwise. ACh-induced currents ranged from 1 to 25 µA at
50 mV.
Characterizing Inhibition by QX-314 and QX-222
The concentrations (IC50) of QX-314 and of QX-222 that inhibited the ACh-induced current by 50% were determined at several holding potentials (see Fig. 4). ACh was applied at 10× its EC50 for each mutant. Leak currents were measured at holding potentials of 50,
125,
75, and
25 mV, each held for 400 ms. ACh
was added and, at the peak of the current, the holding potential
was stepped through the same four values. After a 5-min wash,
QX-222 or QX-314 was applied continuously for 2 min, during
which time leak and ACh-induced currents were determined as
before. This protocol was repeated with increasing concentrations of blocker. We calculated the net ACh-induced currents in
the presence (IACh, QX) and absence (IACh) of blocker, and we fit the
data at each membrane potential to the equation IACh, QX/IACh = 1/{1+([QX]/IC50)}, where [QX] is the blocker concentration.
The IC50 values obtained from the fit were themselves fit to the
Woodhull (1973)
equation: IC50 = IC50(0 mV) exp(
z
F
/RT),
where z is the charge on the blocker, F is the Faraday constant,
is the transmembrane potential difference (inside-outside), and
is the apparent electrical distance from the extracellular medium to the blocker binding site.
|
Protection
The protection of substituted Cys against reaction with MTSEA
afforded by bound blocker was estimated from the second-order rate constants of the reaction in the presence and absence of blocker. The second-order rate constants (k) were determined as previously described (Pascual and Karlin, 1998). Oocytes were superfused for 20 s with ACh (at 10× the EC50 as determined for each mutant), for 3 min with bath solution, for 2-25 s with
MTSEA in the presence of ACh, or for 1-4 min with MTSEA in
the absence of ACh, for 3 min with bath solution, for 20 s with
ACh, and for 3 min with bath solution; this sequence was repeated several times. MTSEA was applied at concentrations ranging from 5 µM for the rapidly reacting residues up to 10 mM for
the slowly reacting residues. MTSEA was applied also in the presence of QX-314 and QX-222, both in the presence and absence
of ACh. The peaks of the ACh-induced currents obtained before
and after the exposure to MTSEA were fit to It = I
+ (I0
I
)
exp(
kxt), where I0 is the initial ACh-induced peak current, It is
the ACh-induced current after a cumulative time, t, of application of MTSEA at concentration, x, and I
is the ACh-induced
current after complete reaction of the Cys. Note that the fraction
of unmodified receptors = (It
I
)/(I0
I
) (Pascual and Karlin, 1998
).
In the presence of ACh, the receptors are distributed among a
number of states: closed, open, and desensitized. During the 2-25 s
that MTSEA and ACh (at 10× EC50) were applied, the receptor was predominantly in the open state or the rapid-onset desensitized state. There was little slow desensitization during this time, and in any case the rate constants for the reactions of two mutants in the slow-onset desensitized state were small (Pascual and
Karlin, 1998; Wilson and Karlin, 1998
). The rate constant for the
reaction in the mixed open state and rapid-onset desensitized
state we call kopen. When blocker was also added, it could have
bound to the open state and to the rapid-onset desensitized state
(Neher, 1983
). To compare the effects of QX-314 and QX-222, at
different concentrations, on the rate constants for the reactions
of MTSEA with the different mutants, we estimated the rate constants for the reactions of MTSEA with the blocked state(s),
kblocked, from the observed rate constants in the presence of
blocker, kobs, and from the IC50 for the particular blocker and
mutant. We assumed that the observed rate constant, kobs, is approximated by kobs = kopen(1
y) + kblockedy, where y, the fraction
of channels that was blocked, is given by: y = 1/(1 + IC50/[QX]).
With each oocyte, we first applied MTSEA (in the presence of
ACh) several times in the absence of blocker to determine kopen, and then applied MTSEA several times in the presence of
blocker to determine kobs. The concentrations of blocker used
were in the range of 2-5× IC50 for the given blocker and mutant,
so that y ranged from 2/3 to 5/6. If we take the extent of protection against reaction to be [1 (kblocked/kopen)], then 1
(kblocked/kopen) = (1/y)[1
(kobs/kopen)]; i.e., the (corrected) extent of protection was 1.2-1.5× the observed extent of protection.
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RESULTS |
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Eight residues in M2 were substituted, one at a time,
by Cys and expressed in Xenopus oocytes together with
wild-type
,
, and
subunits. In each case, the current
elicited by ACh was inhibited by QX-314 and QX-222,
and the inhibition increased with increasing concentration of blocker. This inhibition is illustrated for
L251C
and QX-314 in Fig. 4. We derived the IC50s for the inhibition by the blockers of the ACh-induced currents at
each holding potential from such experiments. The
IC50 for inhibition of the mutants by QX-314 ranged
from 0.4× (
T244C) to 4.6× (
V255C) the IC50 for
wild type (Table I). The range for QX-222 was from 0.05× (
T244C) to 1× (
S252C) the IC50 for wild type.
Typically, when QX-314 and ACh were washed out,
there was an immediate increase in the amplitude of
the current before it returned to the baseline (Fig. 4,
B-D). This increased current resulted either from the blocker dissociating from its site of inhibition before
ACh dissociated from its binding sites or from the
faster dilution below effective concentration of the
blocker (initially at 2-5× IC50) than of ACh (initially at
10× EC50).
|
For both blockers and for all mutants, the more negative the holding potential, the greater the inhibition
of current (Fig. 4). The voltage dependence of the inhibition was consistent with the site of inhibition being
in the open channel. The voltage dependence of the
IC50 was fit by the Woodhull (1973) equation to yield
the blocker charge times the apparent electrical distance, z
. For these quaternary ammonium blockers,
z = 1. For QX-314,
was 0.35 for wild type and ranged
from 0.28 for
V255C to 0.57 for
L251C (Table I). For
QX-222,
was 0.80 for wild type and ranged from 0.34 for
L258C to 0.68 for
T244C and
S248C. The effects of the mutations on the apparent electrical distances for QX-314 and for QX-222 were not correlated.
The interpretation of the apparent electrical distances
is difficult because the IC50s are dependent not only on
the equilibrium dissociation constants of the blockers,
but also on the kinetics of the transitions between functional states.
The reactions of MTSEA with engineered Cys exposed in the channel were manifested by irreversible
effects on the ACh-induced current. We determined
the rates of the irreversible changes in the response to
ACh and the effects of QX-314 and QX-222 on these rates. These blockers had no effect on the rates of reaction of the two Cys-substituted residues closest to the
extracellular end of M2: the reactions of MTSEA in the
presence of ACh with L258C (Fig. 5, A and B) and
E262C were unchanged by the addition of QX-314 or
QX-222 at concentrations several times their IC50s. By
contrast, the reactions of MTSEA in the presence of
ACh with Cys-substituted residues deeper in the channel
were markedly slowed by the addition of these blockers. For example, the irreversible reaction of MTSEA in
the presence of ACh with
S248C was nearly stopped
by the addition of QX-314 (Fig. 5, C and D).
|
QX-314 retarded the reactions of MTSEA only in the
presence of ACh at the two residues on which this was
tested, T244C at the intracellular end of the channel
and
L251C in the middle of the channel. The time
course of the reaction of
T244C with MTSEA in the
presence of ACh, first with, and then without, QX-314 is shown in Fig. 6 A. The rate of reaction was faster after
QX-314 was removed. On the other hand, the rate of
reaction of MTSEA in the absence of ACh was not
changed by the addition of QX-314 (Fig. 6 B). Similar
results were obtained with
L251C.
|
The extent of the retardation of the MTSEA reaction
depended on the concentration of blocker. This is illustrated by the rate constants of the reactions of MTSEA
in the presence of ACh with S248C and
V255C, in
the additional presence of different concentrations of
QX-314 (Fig. 7). To compare the effects of QX-314 and
QX-222 on the rates of reaction of the different mutants, we estimated the rate constants for the reactions
of MTSEA with the blocked state from the observed
rate constants in the presence and absence of blocker
and from the IC50 for the particular blocker and mutant (see METHODS). The IC50 for a particular blocker
and mutant is a function not only of the equilibrium
dissociation constant for the blocker binding to the
open channel, but also of kinetic constants characterizing the isomerizations of the receptor among closed, open, and desensitized states. Although the IC50s (Fig.
7, arrows) were two to four times smaller than the concentrations of blocker giving half of the maximum retardation of rate,2 the use of the IC50 to estimate the
fraction of a given mutant blocked at different concentrations of blocker yielded consistent rate constants for
the blocked state.
|
We take as the extent of protection by blocker, 1 (kblocked/kopen). At the extracellular end of M2,
L258C
and
E262C were not protected by QX-314 or QX-222
(Fig. 8). At the intracellular end of M2,
E241C,
T244C, and
S248C were nearly completely protected. Between the fully protected and the unprotected residues,
L251C was protected ~70% and
V258C ~60% by both blockers.
S252C was protected
58% by QX-222 and not at all by QX-314.
|
The reaction of T244C with an uncharged reagent,
MTSEH, was also retarded by QX-314 in the presence
of ACh: the protection was 88% ± 2% (n = 5). QX-314
had no effect on the rate of the reaction of MTSEH in
the absence of ACh.
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DISCUSSION |
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We found that QX-314 and QX-222, two quaternary
ammonium lidocaine derivatives that act as open-channel blockers, protected channel-lining residues substituted by Cys from reaction with positively charged
MTSEA. The protection was partial from V255C to
L251C and was nearly complete from
S248C to
E241C
(Fig. 8). There are a number of possible mechanisms
for this protection, including steric hindrance, charge
repulsion, and allosteric stabilization of unreactive receptor states.
The last mechanism is not a likely explanation for the
observed protection. The rate constants for the reactions of some of the mutants are quite different in different functional states of the receptor, so that the
blockers could retard the reactions of these mutants by
promoting the less reactive state. Such a mechanism,
however, would affect all of the state-dependent and none of the state-independent reaction rates, contrary
to our results. The rate constants for the reactions of
E241C,
T244C,
L251C, and
V255C with extracellularly applied MTSEA were much larger in the presence of ACh (in the open and rapid-onset desensitized states) than in the absence of ACh (in the predominantly closed state) (Pascual and Karlin, 1998
), and
these mutants were protected by QX-314 and QX-222
(Fig. 8). On the other hand, the rate constants for the
reactions of
S248C and
S252C with MTSEA were not
much different in the presence and absence of ACh,
and these mutants were also protected. Conversely,
L258C and
E262C were not protected, even though
they reacted much faster in the presence of ACh than in its absence. Thus, the blockers did not act by stabilizing an unreactive state from a remote site.
It is also unlikely that the blockers protected the Cys-substituted residues by promotion of the closed state
through direct competition with ACh. Although the
concentrations of QX-314 and QX-222 used to protect
against MTSEA were high enough that some binding to
sites in addition to the open channel was possible (Neher, 1983), there was no protection of
L258C or
E262C, which were far less reactive in the absence
than presence of ACh.
The mechanism of protection also could not have
been solely electrostatic repulsion. QX-314 protected
T244C against the reaction of the neutral reagent
MTSEH. Furthermore, the protection of
S252C by
QX-222 and not by QX-314 indicates that the mechanism of protection was not primarily electrostatic.
The most likely mechanism of protection was the
steric hindrance of access to the substituted Cys by QX-314 and QX-222. Since V255C was the protected residue closest to the extracellular end of M2, this residue
marks the extracellular end of the binding site for the
two blockers. If we compare the dimensions of QX-314 and
M2, configured in the open state as an
helix
(Karlin and Akabas, 1995
), and if the xylyl moiety of
QX-314 overlaps
V255, the triethylammonium group
overlaps
S248 (Fig. 9). QX-314 or QX-222 bound in
this location would protect the residues from
V255C
to
S248C by occluding them directly and would protect
T244C and
E241C by blocking the pathway to
these residues from the extracellular medium.
|
The orientation of the blocker with its positively
charged quaternary ammonium group toward the intracellular end of the channel would be favored by the
electrostatic potential profile in the channel. The net
electrostatic potential at each point in the channel is
the sum of the electrical distance times the transmembrane potential and of the intrinsic potential due to
fixed charges and dipoles in the protein and lumen of
channel. The electrostatic potential in the open channel is ~75 mV more negative at S248 than at
V255
(Pascual and Karlin, 1998
), so that QX-314 or QX-222 would bind ~20× more tightly with its charged ammonium group near
S248 than near
V255. The proposed location and orientation of the blocker in the
channel (Fig. 9) are the same as those proposed by
Charnet et al. (1990)
.
Assuming the location and orientation of the blocker
is as shown in Fig. 9, we can estimate the electrostatic
contribution to the binding energy. The net electrostatic potential in the open channel in the vicinity of
S248, with a transmembrane potential of
50 mV, was
estimated to be ~
100 mV relative to the extracellular medium (Pascual and Karlin, 1998
). Therefore, the
electrostatic contribution to the binding energy per
mole due to the quaternary ammonium group of QX-222 or QX-314 binding close to
S248 would be approximately four RT.
The apparent affinity constant for QX-222 estimated
from burst durations and open times (Neher, 1983)
can be extrapolated to ~2.5 × 104 M
1 at
50 mV. The
affinity of QX-314 is considerably greater than that of
QX-222 (Neher and Steinbach, 1978
). The free energy
per mole of QX-222 binding would be ~10 RT. Thus,
hydrophobic and van der Waals interactions would
contribute approximately six RT to the binding of QX-222 and more to the binding of QX-314. The side
chains of
L251 and
V255, the aligned side chains in
, and possibly the aligned side chains in
and
, are
all more exposed in the open than in the closed channel (Akabas et al., 1994
; Pascual and Karlin, 1998
; Zhang
and Karlin, 1998
). These hydrophobic side chains are
likely to interact with bound blocker (Fig. 9).
Although the mutation of (7)L247 (aligned with
L251) to Thr rendered the neuronal, homopentameric
7 complex insensitive to QX-222 (Revah et al.,
1991
), the mutation in mouse-muscle receptor of the
two
L251 to Cys, and the mutation of the two
V255
to Cys, increased the IC50 for QX-314 but decreased the
IC50 for QX-222 (Table I). The effects of these mutations on IC50 may have been confounded by underlying
effects of the mutations on the kinetics of state transitions.
The patterns of protection indicate that QX-314 and
QX-222 bind at the same depth in the channel. Yet for
wild-type receptor, the electrical distances estimated
from the fits of the IC50s to the Woodhull equation
were quite different, 0.35 for QX-314 and 0.80 for QX-222. The IC50, however, depends not only on the kinetics of binding to the open channel but also on the kinetics of the transitions of the receptor among states.
Also, the IC50 was determined after considerable fast
desensitization had occurred. Thus, the voltage dependence of the IC50 does not have a simple interpretation.
The complexity of the IC50 could partly account for the
variation we observed in electrical distance from 0.28 to
0.57 for QX-314 and from 0.34 to 0.68 for QX-222
among the Cys-substitution mutants. Two different single-channel analyses of QX-222 blocking gave electrical
distances of 0.78 (Neher and Steinbach, 1978) and
~0.4 (Neher, 1983
). Voltage-jump-relaxation analysis
yielded electrical distances from 0.65 to 0.75 for QX-222 (Charnet et al., 1990
) and 0.72 for QX-314 (Horn
and Brodwick, 1980
). In general, quite disparate electrical distances for different ACh receptor channel
blockers have been reported (e.g., Table I in Sanchez
et al., 1986
); whether these reflect different binding sites in the channel or other complications is not
known. The electrical distance estimated by the Woodhull (1973)
equation may not be a consistent marker of
position in the channel.
The protection of S252C by QX-222 and not by QX-314 (Fig. 8) was unexpected and may indicate that the
two blockers do not bind in exactly the same configuration. The orientation of the xylyl ring structure shown
in Fig. 9 is drawn roughly in the plane containing the
two
M2 segments; the M2 segments and
,
, and
subunits are not shown. Because the quaternary ammonium group is symmetrical, the somewhat rigid blocker
molecule can rotate around its axis, which would maintain similar interactions of the ammonium group but
change the subunit interactions of the xylyl ring. Different distributions of axial rotation for the two blockers
could be the basis for the different protection of
S252C. In addition, the rotation of the xylyl group
and the presumed widening of the channel toward the
extracellular end could account for the incompleteness
of the protection of
L251C and
V255C, as well as of
S252C.
In the absence of ACh, QX-314 did not protect
T244C or
L251C.
T244C is the mutant that reacts
most rapidly with MTSEA, both in the presence and absence of ACh, and the inhibition due to the reaction is
complete (Pascual and Karlin, 1998
).
T244C is at the
intracellular end of the channel, so that QX-314 binding above would protect
T244C from extracellular
MTSEA coming through the channel. Thus,
T244C
affords a sensitive test for protection. The lack of protection in the absence of ACh indicates that QX-314 did not bind in the closed channel at the concentrations tested.
The poor binding of QX-314 to the closed channel is
not due to the obstruction of the channel by the activation gate, which we have located between G240 and
T244, at the intracellular end of the channel (Wilson
and Karlin, 1998
). Notwithstanding, there could be a
partial obstruction at the extracellular end of the
closed channel that would limit access by channel
blockers but not by the smaller methanethiosulfonates
used to locate the gate or by inorganic cations. Alternatively, the blockers could bind poorly in the closed
channel because the net electrostatic potential is ~100
mV less negative in the closed than in the open channel (Pascual and Karlin, 1998
), and the side chains of
L251 and
V255 and of the aligned residues in the
other subunits are less exposed (Akabas et al., 1994
;
Pascual and Karlin, 1998
; Zhang and Karlin, 1998
). Restating this, we suggest that more favorable electrostatic
and hydrophobic interactions, rather than greater accessibility, could account for the stronger binding of
blockers in the open than in the closed channel. Some
of the complexities of local anesthetic action on the
voltage-gated Na+ channel have been explained on the
basis of different intrinsic local-anesthetic binding affinities in different states (the modulated receptor
model); however, in contrast to the ACh receptor, in the Na+ channel, accessibility to the binding site controlled by the activation and inactivation gates is crucial
for the binding of quaternary ammonium derivatives of
local anesthetics like QX-314 (Hille, 1977
; Ragsdale et
al., 1994
).
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FOOTNOTES |
---|
Address correspondence to Arthur Karlin, Center for Molecular Recognition, Columbia University, 630 West 168th Street, Box 7, New York, NY 10032. Fax: 212-305-5594; E-mail: ak12{at}columbia.edu
Original version received 29 June 1998 and accepted version received 3 September 1998.
This research was supported by research grants to A. Karlin from the National Institutes of Health (NIH) (NS-07065), the Muscular Dystrophy Association, and the McKnight Endowment Fund for Neuroscience. During part of this work, J.M. Pascual was supported by NIH Neurological Sciences Academic Development Award NS01698. We thank Dr. Rune Sandberg of Astra for gifts of QX-222 and QX-314.We thank Drs. Myles Akabas, Jonathan Javitch, and Gary Wilson for comments on the manuscript. We are also grateful to Gilda Salazar-Jimenez for oocyte culture.
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Abbreviations used in this paper |
---|
ACh, acetylcholine; M2, second membrane-spanning segment; MTSEA, 2-aminoethyl methanethiosulfonate; MTSEH, 2-hydroxyethyl ethanethiosulfonate; NCI, noncompetitive inhibitor.
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