Departments of 1 Pharmacology and 2 Cell and Molecular Physiology, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599; and 3 Division of Infectious Diseases and International Health, Duke University, Durham, North Carolina 27710
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ABSTRACT |
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Neuronal
7 nicotinic acetylcholine receptors (nAChRs) are
permeable to Ca2+ and other divalent cations. We
characterized the modulation of the pharmacological properties of
nondesensitizing mutant (L247T and
S240T/L247T)
7 nAChRs by
permeant (Ca2+, Ba2+, and Sr2+) and
impermeant (Cd2+ and Zn2+) divalent cations.
7 receptors were expressed in Xenopus oocytes and studied with two-electrode voltage clamp. Extracellular permeant divalent cations increased the potency and maximal efficacy of ACh,
whereas impermeant divalent cations decreased potency and maximal
efficacy. The antagonist dihydro-
-erythroidine (DH
E) was a strong
partial agonist of L247T and
S240T/L247T
7 receptors in the
presence of divalent cations but was a weak partial agonist in the
presence of impermeant divalent cations. Mutation of the
"intermediate ring" glutamates (E237A) in
L247T
7 nAChRs eliminated Ca2+
conductance but did not alter the Ca2+-dependent increase
in ACh potency, suggesting that site(s) required for modulation are on
the extracellular side of the intermediate ring. The difference between
permeant and impermeant divalent cations suggests that sites within the
pore are important for modulation by divalent cations.
acetylcholine receptor; calcium; M2 domain; potency; permeation
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INTRODUCTION |
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NEURONAL
NICOTINIC ACh receptors (nAChRs) are members of the
Cys-loop superfamily of ligand-gated ion channels that also includes GABAA receptors, serotonin type 3 receptors, glycine
receptors, and an invertebrate glutamate-gated Cl channel
(25). Like muscle-type nAChRs, neuronal nAChRs are formed
as pentameric complexes of subunits arranged around a central ion-permeable pore (13). To date, nine neuronal nAChR
-subunits (
2-
10) and three
neuronal nAChR
-subunits (
2-
4)
have been cloned (6, 17). Functional ion channels can be
formed from various combinations of these subunits (32).
In particular, nAChR
7-subunits can form functional,
homomeric ion channels when expressed in Xenopus oocytes
(32) or in vitro (30).
Extracellular Ca2+, which can vary dramatically in
concentration during high synaptic activity (8), has been
shown to modulate the activity of neuronal nAChRs (9, 16, 18, 19,
27, 33, 42). For example, nicotinic responses of medial habenula neurons are rapidly increased when extracellular Ca2+ is
raised from ~10 µM to 4 mM even though unitary currents through the
receptors are decreased, indicating an increase in channel activity
(33). Neuronal 3
4 nAChRs
expressed in Xenopus oocytes (42) and
4
2 nAChRs expressed in HEK 293 cells
(9) show similar Ca2+-dependent increases in
channel activity and decreases in unitary current amplitudes. The
potencies of agonists on oocyte-expressed
7 nAChRs are
also increased by the presence of extracellular Ca2+
(16, 19), perhaps due to Ca2+ binding to a
region on the extracellular NH2-terminal domain (16,
22). These increases in potency are likely due to alterations in
channel gating rather than enhanced ligand binding, because Ca2+ inhibits the binding of ACh to muscle-type nAChRs
(11).
7 nAChRs are highly permeable to Ca2+
(10, 21, 39, 40). Ca2+ influx through
presynaptic
7 nAChRs modulates the release of neurotransmitters in the central and peripheral nervous systems (31).
7 receptors are also permeable to
Ba2+ and Sr2+ (39) but are blocked
by extracellular Zn2+ (37).
In this study, we determined whether permeant divalent cations
(Ca2+, Ba2+, or Sr2+) modulate the
maximal efficacy and/or agonist potency of 7 nAChRs differently than impermeant or blocking divalent cations
(Cd2+ or Zn2+). Because the rapid
desensitization of wild-type
7 nAChRs (14) introduced errors in the measurement of peak currents, we studied slowly desensitizing receptors containing a leucine-to-threonine mutation at position 247 (L247T) in the pore-lining M2
domain (38). These receptors have an EC50 for
ACh ~100-fold lower than that of wild-type
7 nAChRs and are activated by several antagonists of wild-type
7
nAChRs, including (+)-tubocurarine, dihydro-
-erythroidine (DH
E),
and hexamethonium (2). To explain these results, one model
proposes that mutation of L247 confers conductance to one
of the desensitized states (2). Other models suggest that
mutations of L247 alter channel gating to favor the open
state (20, 26, 36). We report that L247T
7 nAChRs and receptors with two mutations in the M2
domain (S240T/L247T) that also desensitize
slowly (30) were permeable to Ca2+,
Ba2+, and Sr2+ but were blocked by
Cd2+. Permeant divalent cations caused a significant
increase in ACh potency, whereas impermeant divalent cations decreased
ACh potency. In addition, we show that the activation of mutant
7 nAChRs by DH
E depended critically on the presence
of permeant divalent cations. Mutation of the "intermediate ring"
glutamates (E237), which eliminates Ca2+
permeation (4), did not eliminate
Ca2+-dependent changes in agonist potency, suggesting that
the site required for this modulation was on the extracellular side of E237. These results suggest that occupancy by permeant
divalent cations of site(s) in the pore may participate in the
regulation of channel activity and agonist potency. Alternatively,
extracellular sites that have been identified as being important for
the modulation of the receptor by Ca2+ (22)
may have ion selectivity properties that are similar to those of the
pore. Preliminary accounts of these results have appeared in abstract
form (15, 28).
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METHODS |
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Chemicals.
Divalent cation salts were obtained from Fluka. DHE was obtained
from RBI (Natick, MA). Gentamicin was obtained from GIBCO BRL
(Gaithersburg, MD). ACh, ethylene glycol-bis(
-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA), and
1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid
(BAPTA) were obtained from Sigma (St. Louis, MO).
Site-directed mutagenesis.
Chick wild-type 7 nAChR cDNA (generously provided by M. Ballivet), subcloned into the pAMV vector (34), and the
S240T/L247T mutant (generated with the
Clontech Transformer kit; Clontech, Palo Alto, CA) were gifts from C. Labarca (California Institute of Technology). L247T and
E237A/L247T
7 nAChR mutations
were generated from chick wild-type
7 cDNA by
four-primer PCR with Pfu DNA polymerase (Stratagene, La
Jolla, CA). All mutations were confirmed by DNA sequencing.
Microinjection of cRNA and maintenance of Xenopus oocytes.
The 7 cDNAs were digested with NotI to
generate linear templates. Capped cRNA was transcribed in vitro with T7
RNA polymerase by using the mMessage mMachine kit (Ambion, Austin, TX)
according to the manufacturer's instructions. cRNA was resuspended in
100 mM KCl or water. Female Xenopus laevis (Nasco, Atkinson,
WI) were fully anesthetized in 0.2% tricaine (3-aminobenzoic acid
ethyl ester), and oocytes were surgically removed. Each frog was used a
maximum of four times, in accordance with University of North Carolina
at Chapel Hill Institutional Animal Care and Use Committee guidelines.
Oocytes were treated with collagenase (Sigma) to remove the follicular
cell layer (23). Oocytes were injected with 20 ng of cRNA
and incubated at 19°C for 2-5 days in ND96 (in mM: 96 NaCl, 2 KCl, 1 MgCl2, 1.8 CaCl2, and 5 Na-HEPES, pH
7.5) supplemented with 50 µg/ml gentamicin and 0.55 mg/ml sodium pyruvate.
Solutions.
To measure currents carried by divalent cations, oocytes were
superfused with 90 mM N-methyl-D-glucamine
methanesulfonate (NMG-MeS), 10 mM HEPES, pH 7.2-7.4, with 10 mM of
one of the following: Ba(OH)2, Ca2+-gluconate,
Sr(OH)2, MgSO4, or CdSO4. To
determine ACh and DHE dose-response relationships and to measure
spontaneous receptor activity in the absence of agonist, oocytes were
superfused with normal extracellular solutions (in mM: 96 NaCl, 2 KC1,
1 MgCl2, 10 HEPES, pH 7.5) plus (in mM) 2.5 CaCl2, 2.5 BaCl2, 2.5 SrCl2, 1 CdCl2, 1 ZnCl2, or 1 EGTA.
Two-electrode voltage clamp.
To minimize the activation of Ca2+-activated
Cl channels, all oocytes were injected with 46 nl of 50 or 100 mM BAPTA (~5-10 mM final intracellular concentration) 15 min before recording (42). The effectiveness of BAPTA was
confirmed by comparing current-voltage (I-V) relationships
obtained in Ca2+-containing normal extracellular solution
to I-V curves obtained in Ca2+-free
extracellular solution containing 1 mM EGTA. In Ca2+-free
extracellular solution, I-V curves displayed strong inward rectification. In Ca2+-containing extracellular solution in
the absence of BAPTA injection, I-V curves were nearly
linear because of outward current carried by the
Ca2+-activated Cl
channels at positive
voltages. In Ca2+-containing extracellular solution after
BAPTA injection, I-V curves showed strong inward
rectification and were indistinguishable from those recorded in the
absence of extracellular Ca2+. Thus strong inward
rectification in Ca2+-containing extracellular solution was
taken as evidence that the injected BAPTA was effective in chelating
intracellular Ca2+. When currents carried by divalent
cations alone were measured, Ca2+-activated
Cl
current was further reduced by replacing
Cl
in the bathing solutions with MeS (40).
When cells were bathed with Cl
-free solutions, the bath
ground electrode was placed in a chamber containing 3 M KCl and
connected to the bath by a KCl-filled salt bridge.
Analysis of dose-response curves. Dose-response relationships were fitted to the Hill equation with Prism software (GraphPad Software, San Diego, CA). To control for rundown during the acquisition of dose-response data, the test responses were normalized to the peak currents from repeated applications of a standard dose of ACh. An F-test was performed to determine whether there were statistically significant differences in the EC50 values determined under different conditions.
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RESULTS |
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Responses of wild-type, L247T, and
S240T/L247T 7 nAChRs.
Figure 1A shows traces of
ACh-evoked currents of chick wild-type, L247T, and
S240T/L247T
7 nAChRs expressed
in Xenopus oocytes and recorded in normal extracellular
solutions containing 1 mM EGTA or 2.5 mM Ca2+. Wild-type
7 nAChRs activated rapidly, desensitized completely within 5 s, and required high concentrations of ACh for full
activation (Fig. 1A; Refs. 14, 38,
and 40). In contrast, L247T (38) and
S240T/L247T (30)
7
receptors showed very slow desensitization during a 30-s application of
ACh (Fig. 1, B and C). The desensitization kinetics of L247T and S240T/L247T
7 receptors were similar in the absence and presence of
Ca2+. All three receptor types showed a
Ca2+-dependent increase in maximal current amplitudes
(i.e., the responses evoked by a maximal concentration of ACh). We
elected to use L247T and
S240T/L247T
7 nAChRs to further
evaluate the effect of divalent cations because their slow
desensitization properties allowed us to make more accurate
measurements of the peak evoked currents.
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ACh-evoked inward currents are carried by
Ca2+, Ba2+,
Mg2+, and
Sr2+ but not Cd2+.
To determine whether L247T and
S240T/L247T 7 nAChRs are
permeable to divalent alkaline earth and transition metal cations, we
recorded ACh-evoked responses in NMG-MeS solutions containing 10 mM
Ca2+, Ba2+, Mg2+, Sr2+,
or Cd2+. These solutions contained the impermeant cation
NMG+ (90 mM) instead of Na+ or K+,
so inward currents could only be carried by the divalent cations. Efflux of Cl
through endogenous
Ca2+-activated Cl
channels was prevented by
injecting the oocytes with BAPTA and by replacing extracellular
Cl
with MeS
. We could not measure currents
carried by Zn2+, because NMG-MeS solutions containing ~1
mM zinc acetate, Zn(OH)2, or ZnS04 formed an
insoluble precipitate. However, 1 mM Zn2+ was previously
shown to block wild-type and L247T
7 nAChRs
(37). We compared the amplitudes of the currents carried
by divalent cations with those recorded in 96 mM NaCl (normal
extracellular solution) or a solution containing no permeant ions (96 mM NMG-MeS).
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Ca2+,
Ba2+, and
Sr2+ increase potency of ACh on mutant
7 nAChRs.
To evaluate the effect of permeant and impermeant divalent cations on
the potency of ACh, dose-response curves were compared in normal
extracellular solutions containing 2.5 mM Ca2+,
Ba2+, or Sr2+ or 1 mM Cd2+,
Zn2+, or EGTA (Fig. 3; Table
1). On L247T receptors,
ACh had much higher potency and slightly higher efficacy in the
presence of permeant divalent cations than in impermeant divalent
cations or EGTA (Fig. 3A). Specifically, ACh dose-response curves obtained in solutions containing Ca2+,
Ba2+, or Sr2+ had EC50 values that
were ~10-fold lower than those in EGTA (Table 1; P < 0.001, F-test). Cd2+ and Zn2+,
however, decreased the potency of ACh (Table 1; P < 0.001). The maximal efficacy of ACh was significantly higher in the
presence of Sr2+ than in EGTA (P < 0.05, 1-way ANOVA), but the differences in maximal efficacy in
Ca2+ and Ba2+ did not reach statistical
significance. The maximal efficacies in Cd2+ and
Zn2+ were significantly lower than in EGTA
(P < 0.001). The presence or absence of
Mg2+ had no effect on ACh dose-response curves of
L247T
7 nAChRs (not shown).
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Spontaneous activity of L247T and
S240T/L247T 7 nAChRs was
increased by Ca2+,
Ba2+, and
Sr2+ but not by
Cd2+ or
Zn2+.
L247T
7 receptors have a detectable level of
activity in the absence of agonist (3) that is evident as
an inhibition of basal current by methyllycaconitine (MLA), a potent
inhibitor of
7 receptors (35, 43). To
determine whether this agonist-independent activity also depended on
the presence of permeant divalent cations in the external solutions, we
tested the inhibition by MLA of basal current of oocytes expressing
L247T and S240T/L247T
7 receptors. Figure 4
shows that the MLA-sensitive basal current of both mutant receptors was
significantly higher in the presence of Ca2+,
Ba2+, or Sr2+ than in external solutions
containing Cd2+, Zn2+, or EGTA. The basal
activity of L247T receptors was significantly greater than
that of S240T/L247T receptors, congruent with
the higher potency of ACh on L247T receptors than
S240T/L247T receptors (Fig. 3; Table 1). These
results indicate that in addition to increasing the potency of ACh and
increasing the maximal efficacy of L247T and
S240T/L247T
7 nAChRs, permeant
divalent cations potentiate the agonist-independent spontaneous
activity of these receptors.
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Ca2+ dependence of increase in ACh
potency and efficacy.
To further characterize the interaction between permeant divalent
cations and the site responsible for the modulation of agonist potency
and efficacy, we studied the effects of different concentrations of
extracellular Ca2+ on the ACh dose-response
characteristics. Figure 5 shows a plot of
the EC50 of ACh dose-response curves vs. the concentration of Ca2+ in the external solution. For the L247T
7 nAChR, the concentration of Ca2+ needed to
cause a half-maximal increase in ACh potency was ~0.1 mM. We also
compared ACh dose responses in the presence of 0.3 mM Ca2+,
Ba2+, or Sr2+, where differences between the
EC50 values would be most apparent. However, the ACh
EC50 values were indistinguishable (0.88 ± 0.086, 0.74 ± 0.094, and 0.90 ± 0.13 µM in Ca2+,
Ba2+, and Sr2+, respectively; n = 7-11, data not shown).
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DHE is a strong agonist of L247T
7
nAChRs (and a partial agonist of S240T/L247T
7 nAChRs) only in the presence of permeant divalent
cations.
DH
E is an antagonist of neuronal nAChRs, but it evokes responses of
L247T
7 nAChRs (2). To
determine whether the activation of L247T and
S240T/L247T
7 receptors by
antagonists was also sensitive to permeant and impermeant divalent
cations, we tested the activation of the receptors by DH
E in the
presence of Ca2+, Ba2+, Sr2+,
Cd2+, Zn2+, and EGTA (Fig.
6). In each cell, the responses to DH
E
were normalized to the maximal ACh-evoked response in an external
solution containing 2.5 mM Ca2+. On L247T
receptors (Fig. 6A), DH
E had higher potency and much
higher efficacy in the presence of permeant divalent cations than in the presence of impermeant divalent cations or EGTA. Specifically, in
extracellular solutions containing Ca2+, Ba2+,
or Sr2+, DH
E had maximal efficacies of 95%, 100%, and
96%, respectively, relative to 10 µM ACh. The kinetics of activation
and desensitization of DH
E- and ACh-evoked responses were the same
(data not shown). In solutions containing EGTA, Cd2+, or
Zn2+, DH
E had a maximal efficacy of only 15%, 7%, and
6%, respectively.
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Elimination of Ca2+ permeation by
mutation of E237 does not prevent modulation.
Because only permeant divalent cations augmented the pharmacological
responses of the mutant 7 nAChRs, we hypothesized that modulatory site(s) could be present within the channel pore or at an
intracellular site near the pore where divalent cation concentrations could reach millimolar concentrations when the receptors are activated. These sites could facilitate or amplify the effects of the modulatory sites in the NH2-terminal extracellular domain (16,
22). To investigate one possible intracellular site, we
constructed an E237A/L247T
7
nAChR that contained the glutamate-to-alanine mutation at the
intermediate ring that eliminates Ca2+ permeability
(4). E237A/L247T
7
nAChRs did not conduct inward current in NMG-MeS solutions containing
10 mM Ca2+ as the only permeant ion, even though they
produced robust currents in normal extracellular solutions, confirming
that the receptors were Ca2+ impermeable (not shown).
Nevertheless, ACh dose-response curves of
E237A/L247T
7 nAChRs (Fig.
7) retained the ~10-fold increase in
ACh potency in the presence of Ca2+ compared with EGTA. The
observed decrease in maximal efficacy probably resulted from block of
the Ca2+-impermeable receptors by extracellular
Ca2+. This is supported by the observations that
extracellular Ca2+ (1-10 mM) reduced the amplitude of
inward Na+ current in a dose-dependent manner, and, in the
absence of extracellular permeant ions, extracellular Ca2+
(1-10 mM) reduced the amplitude of outward K+ currents
(data not shown). Because E237A/L247T
7 nAChRs showed an increase in ACh potency despite the
lack of Ca2+ conductance, it is likely that the modulatory
site for permeant divalent cation is on the extracellular side of
position 237.
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DISCUSSION |
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We have shown that the potency and maximal efficacy of agonists of
nondesensitizing 7 nAChRs, and the level of spontaneous agonist-independent activity, were significantly greater in the presence of permeant alkaline earth metal cations (Ca2+,
Ba2+, and Sr2+) than in their absence.
Activation of the receptors by the classic antagonist DH
E depended
critically on the presence of permeating divalent cations. In the
absence of Ca2+, Ba2+, or Sr2+,
DH
E was an antagonist or a very weak partial agonist of the mutant
receptors, whereas in the presence of these ions, DH
E acted as a
strong partial agonist. The presence of Ca2+,
Ba2+, or Sr2+ was required for the activation
of S240T/L247T
7 receptors by
DH
E.
Although the L247T and S240T/L247T
7 nAChRs have very different desensitization kinetics
and ACh EC50 values that are significantly lower than
wild-type
7 nAChRs, there are several important
similarities. Both wild-type and mutant receptors show a decrease in
ACh EC50 values in the presence of Ca2+ (Fig.
3; Ref. 16). Both wild-type and mutant receptors show a
Ca2+-dependent increase in the maximal efficacy of ACh,
although the magnitude of the increase is noticeably smaller for mutant
receptors than for wild-type receptors (Figs. 1 and 3). The small
increase in maximal efficacy of ACh on mutant receptors is probably
because ACh is such a potent agonist of the mutant receptors with high intrinsic efficacy compared with the wild-type receptors
(12). Congruent with this idea, the maximal efficacy of
the partial agonist DH
E on the mutant receptors is significantly
increased in the presence of permeant divalent cations (Fig. 6).
Although DH
E appears to be an antagonist of wild-type receptors and
a weak or strong partial agonist of the mutant receptors (in the absence or presence of permeant divalent cations, respectively), other
"antagonists" of native nicotinic receptors have been shown to be
weak partial agonists of some receptor subtypes (41). Thus
partial agonist behavior of antagonists on the mutant receptors is a
property that has been seen in native nAChRs. Finally, even though the
mutations are in the pore-lining M2 domain, the permeation pathways of
S240T/L247T and wild-type
7
nAChRs are functionally very similar (as measured by single-channel
conductance, inward rectification, ion selectivity, Ca2+
permeability, and voltage-dependent block by QX-222, a quaternary ammonium compound; Ref. 29). Overall, these similarities
suggest that conclusions drawn from the L247T and
S240T/L247T
7 nAChRs are likely
to be valid for wild-type
7 receptors also.
Location of cation binding site required for modulation.
Mutation of E237, an amino acid that forms the intermediate
ring of negative charge that is required for Ca2+
permeation (4), failed to abolish
Ca2+-dependent increase in ACh potency, suggesting that an
intracellular binding site is not required for modulation by permeant
divalent cations, in agreement with previous reports (27,
33). One divalent cation binding site required for the observed
changes in agonist potency and the activation by DHE could be in the NH2-terminal extracellular domain (16, 22).
However, the observed differences between permeant and impermeant
divalent cations support the hypothesis that occupancy of site(s) in
the pore could also be partially responsible. These site(s) could
include the pore's selectivity filter itself or site(s) on the
extracellular side of the intermediate ring glutamates. If the site(s)
were in the transmembrane electric field, one would expect some voltage
dependence of the effect of permeant divalent cations. However, in the
presence of 2.5 mM Ca2+, ACh dose responses of both
nondesensitizing receptors were the same at
60 and
100 mV (data not
shown), suggesting that modulatory site(s), if within the pore, are not
in the electric field. This could be explained if the electric field
was constrained to a section of the pore. Additionally, the site(s)
could be located in the outer vestibule, especially at the region where
the outer vestibule meets the transmembrane portion of the permeation
path (7). It is also possible that extracellular
modulatory sites (16, 22) that may not be in the
permeation pathway have ion selectivity properties similar to those of
the pore or that pore sites and extracellular sites are both required
for the modulatory effects of permeant divalent cations.
Possible mechanisms of divalent cation modulation.
Several mechanisms may explain the effect of permeating divalent
cations on the pharmacological properties of 7 nAChRs.
First, Ca2+, Ba2+, and Sr2+ could
increase the affinity between ACh and the ligand-binding site.
Extracellular Ca2+ levels are reported to alter the
cooperativity of ACh binding in receptors that generate type 1A
currents (believed to arise from
7 nAChRs) in cultured
rat hippocampal neurons (5). However, we found that the
Hill coefficients were nearly the same in the presence of EGTA or
Ca2+, Ba2+, or Sr2+ (Fig. 3; Table
1), suggesting that there is no change in cooperativity in this system.
Furthermore, Ca2+ is a competitive inhibitor of ACh binding
to Torpedo nAChRs in the 0.1-1 mM range
(11), suggesting that increasing extracellular Ca2+ to 2.5 mM would decrease (not increase) ACh occupancy.
Finally, increasing the affinity with which a ligand binds to a
receptor site cannot explain how the receptor can be activated by an antagonist.
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ACKNOWLEDGEMENTS |
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We thank Susan Holley for excellent technical assistance; Mark
Jezyk, Daphne Best, Michael Casey, and Sarah Tanguay for experimental data that contributed to this project; and Ed Westhead for comments on
the manuscript. We also thank M. Ballivet (University of Geneva) for
the 7 nAChR cDNA and C. Labarca and H. A. Lester
(California Institute of Technology) for the
S240T/L247T
7 nAChR cDNA and the
pAMV vector.
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FOOTNOTES |
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This work was supported by National Institute of Neurological Disorders and Stroke Grant NS-37317.
Present addresses: S. Desai, National Institute Allergy and Infectious Diseases, Bethesda, MD 20892; J. W. Lee, Memorial Sloan-Kettering Cancer Center, New York, NY 10021.
Address for reprint requests and other correspondence: R. L. Rosenberg, Dept. of Pharmacology, CB# 7365, Univ. of North Carolina at Chapel Hill, Chapel Hill, NC 27599-7365 (E-mail: bobr{at}med.unc.edu).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
10.1152/ajpcell.00453.2001
Received 21 September 2001; accepted in final form 9 November 2001.
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