Departments of 1 Pharmacology and 2 Cell and Molecular Physiology, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599
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ABSTRACT |
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Neuronal 7 nicotinic ACh
receptors (nAChRs) are permeable to and modulated by Ca2+,
Ba2+, and Sr2+. These permeant divalent cations
interact with slowly desensitizing L247T
7
nAChRs to increase the potency and maximal efficacy of ACh, increase
the efficacy of dihydro-
-erythroidine (DH
E), and increase agonist-independent activity. Mutation of glutamate 172 (E172) to glutamine or cysteine eliminated these effects of
permeant divalent cations. 2-(Trimethylammonium)ethyl
methanethiosulfonate (MTSET), a cysteine-modifying reagent directed
at water-accessible thiols, inhibited ACh-evoked currents of
E172C/L247T
7 nAChRs by >90%,
demonstrating that E172 was accessible to permeant
ions. The data are consistent with a model of
7
receptors, derived from the crystal structure of the ACh binding
protein (AChBP) from Lymnaea stagnalis, in which E172 projects toward the lumen of the extracellular
vestibule. The observations that E172 was essential for
divalent cation modulation of L247T
7 nAChRs
and was accessible to permeating ions suggest that this residue
participates in coupling ion permeation with modulation of receptor activity.
acetylcholine receptor; calcium; Xenopus oocyte; cysteine modification
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INTRODUCTION |
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THE POTENT PSYCHOLOGICAL
EFFECTS of nicotine underscore the importance of neuronal
nicotinic acetylcholine receptors (nAChRs) in the central nervous
system (CNS) (11, 14, 21, 26, 29, 43). Neuronal nAChRs are
ligand-gated ion channels formed by heteromeric complexes of - and
-subunits or by homomeric assemblies of
-subunits. The most
prevalent example of homomeric assembly is the homopentamer of
7-subunits (7, 12, 36). The functional expression of
7 nAChRs in Xenopus oocytes
does not require coexpression of other
- or
-subunits, although
7-subunits may coassemble with other subunits in vivo
(45).
Neuronal 7 nAChRs are widely distributed in the CNS and
serve several different functions (7, 21). They mediate
postsynaptic responses of hippocampal interneurons and other neurons
(2, 18, 21, 22, 31). They modulate the release of
excitatory and inhibitory neurotransmitters in the hippocampus and
other regions (1, 30, 34). They are important in the early
development and proliferation of neurons (34). In
addition,
7 nAChRs promote cell survival in several
experimental models of neurological damage, including hypoxia
(40),
-amyloid exposure (24), deprivation of nerve growth factor (NGF) (27), and
N-methyl-D-aspartate (NMDA) receptor-mediated
excitotoxicity (13). The high Ca2+
permeability of
7 nAChRs (8, 28, 37, 41,
42) is likely to play a role in many of their functions.
In addition to Ca2+, 7 nAChRs are highly
permeable to Ba2+ and Sr2+ (15,
35). These permeant divalent cations modulate the activity of
wild-type and L247T
7 nAChRs by increasing
the potency and maximal efficacy of ACh (15, 19). In
addition, the antagonist dihydro-
-erythroidine (DH
E)
(11) is a strong partial agonist of L247T
7 nAChRs in the presence but not the absence of permeant
divalent cations (15). Mutation of E237,
located at the intracellular end of the transmembrane pore of
7 nAChRs, eliminates Ca2+ influx
(4) but does not alter Ca2+-dependent
increases in ACh potency (15). This suggests that the
site(s) required for modulation by extracellular Ca2+ is in
the extracellular or pore-lining regions. Because permeant but not
impermeant divalent cations modulate L247T
7
nAChRs (15), it is possible that sites necessary for
modulation of
7 nAChRs by divalent cations are in the
ion permeation pathway.
Another site shown to play a role in the modulatory effects of
Ca2+ on 7 receptors is glutamate 172 (E172), located in the extracellular
NH2-terminal domain (19). In
7-V201-5-HT3 receptors (a chimeric
receptor consisting of the NH2-terminal extracellular
domain of
7 nAChRs and the transmembrane and
COOH-terminal domains of 5-HT3 receptors), mutation of
E172 to glutamine (E172Q) abolishes
Ca2+-induced increases in ACh potency and maximal efficacy
(19).
The crystal structure of the ACh binding protein (AChBP), a soluble
protein secreted by glial cells of the snail Lymnaea
stagnalis (6), provides further insight into the role
of E172 in modulating the activity of 7
nAChRs. Computational mapping of the
7 nAChR sequence
onto the crystal structure of the AChBP can be used to generate a model
of the extracellular domain of
7 receptors. In this
model, E172 lines the "bottom" of the outer vestibule,
near the interface of the extracellular NH2 terminus and
the transmembrane domains. Thus the model suggests that
E172 is accessible to permeant divalent cations in the
water-filled vestibule.
In this study, we characterized the role of E172 in the
divalent cation permeation and modulation of L247T
7 nAChRs. We used the L247T
7
receptor (3, 33) as the "background" for the
introduction of E172 mutations because its high potency,
high efficacy, slow desensitization, and large
Ca2+-dependent shifts in potency (15)
increased the resolution of effects of mutations and ionic conditions.
We report that the potency and efficacy of ACh on
7
receptors containing both E172Q and L247T
mutations (E172Q/L247T
7 nAChRs)
were unchanged in the presence of permeable divalent cations
(Ca2+, Ba2+, and Sr2+). In
addition, the E172Q mutation eliminated the high level of
agonist-independent activity of L247T
7
nAChRs (5) and abolished the agonist behavior of the
antagonist DH
E on these receptors (3). We show that
E172 was accessible to permeant ions, as evidenced by the
inhibition of E172C/L247T
7
nAChRs by 2-(trimethylammonium)ethyl methanethiosulfonate (MTSET), a
cysteine-modifying reagent directed at water-accessible thiols
(23). Thus E172 was essential for the
modulation of L247T
7 nAChRs by divalent
cations and was accessible to extracellular ions, suggesting that it is
part of a mechanism that couples ion permeation with modulation of
receptor gating.
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METHODS |
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Chemicals.
Divalent cation salts were obtained from Fluka. DHE was from RBI
(Natick, MA). Gentamicin was from GIBCO-BRL (Gaithersburg, MD).
Methanethiosulfonate (MTS) reagents were from Toronto Research Chemical (Toronto, ON, Canada). All other reagents were from Sigma.
Mutagenesis.
Chick wild-type and L247T 7 nAChR cDNAs were
obtained as previously described (15). E172Q
and E172C mutations were introduced into L247T
7 receptors with the QuickChange method (Stratagene).
All mutations were verified by DNA sequencing.
Oocyte expression. Oocytes were removed surgically and dissociated enzymatically following standard protocols (20, 38) as described previously (15). cRNA was transcribed in vitro with the T7 RNA polymerase using the mMessage mMachine kit from Ambion. Oocytes were injected with 20 ng of cRNA and incubated for 2-3 days at 18°C.
Electrophysiology.
Currents were recorded under two-microelectrode voltage clamp
(GeneClamp and pCLAMP6, Axon Instruments). Typically, oocytes were
bathed in a normal extracellular solution (in mM: 96 NaCl, 2 KCl, 1 MgCl2, 10 HEPES, pH 7.4) containing 1 mM EGTA (ES-EGTA), 2.5 mM CaCl2 (ES-Ca2+), 2.5 mM
BaCl2 (ES-Ba2+), or 2.5 mM SrCl2
(ES-Sr2+). Solution flow was continuous throughout an
experiment (~4 ml/min in a chamber of ~100 µl). To prevent
activation of the endogenous Ca2+-activated
Cl channels, the oocytes were injected with 46 nl of
50-100 mM BAPTA 15-60 min before recording, as described
previously (15). Oocytes were usually held at a voltage of
60 mV, and responses were measured during superfusion with ACh.
Oocytes were rinsed in the extracellular solutions for 3-4 min
between ACh applications.
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 the data, responses were normalized to the
peak currents from repeated applications of a standard dose of ACh (50 and 30 µM for E172Q/L247T and
E172C/L247T 7 receptors,
respectively). An F-test was performed to determine whether
there were statistically significant differences in the EC50 values determined under different conditions. Data
plotted and values reported are means ± SE. Statistical
significance was assumed at P < 0.05.
Structural model of 7.
A model of the chick
7 nicotinic receptor was based on
the coordinates of the AChBP (6). The BIOINBGU
fold-recognition server (http://www.cs.bgu.ac.il/~bioinbgu/; Ref.
17) identified the monomer of the AChBP homopentamer (PDB
ID 1I9B.pdb) as a structure compatible with the chick
7
sequence. A model of the
7 monomer was based on chain A
of the AChBP pentamer by using the Modeler module of the InsightII
system (www.accelrys.com). A homopentamer of
7 was
generated by superimposing five different copies of the
7 homology model on each of the monomers in the AChBP
homopentamer. The Profiles-3D module of the InsightII molecular modeling system was used to evaluate the compatibility of the model of
the
7 pentamer with the
7 sequence. The
imaging program SPOCK (http://mackerel.tamu.edu/spock/) was used to
create figures of the homology model.
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RESULTS |
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ACh-evoked responses of E172Q/L247T are
slowly desensitizing.
Figure 1 shows some of the key
functional properties of the E172Q/L247T
7 receptor. Figure 1A shows that
E172Q/L247T
7 nAChRs, like
L247T
7 receptors (33),
displayed slow desensitization kinetics. Prolonged application of ACh
produced sustained responses similar to those of the L247T
7 receptor. ACh dose-response curves (Fig.
1B) reveal that the EC50 of
E172Q/L247T
7 receptors fell
between those of L247T
7 nAChRs and
wild-type
7 receptors. In the presence of 2.5 mM
Ca2+, the EC50s for wild-type,
E172Q/L247T, and L247T receptors
were 180, 38, and 0.34 µM, respectively (see also Refs.
12, 15, 33). The results show
that the slow desensitization of L247T
7
receptors was maintained in E172Q/L247T
7 nAChRs even though E172Q/L247T
7 receptors did not display the extremely low
EC50 for ACh that was seen for L247T
7 receptors (33).
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Ca2+,
Ba2+, and
Sr2+ do not stimulate
E172Q/L247T 7 nAChRs.
Previous reports demonstrated that permeant divalent cations modulate
agonist responses of L247T
7 ACh receptors
(15, 19). In the presence of permeant divalent cations,
ACh activates the L247T
7 receptor with an
EC50 that is ~10-fold lower than in the presence of EGTA
(15). In addition, the maximal efficacy of ACh is
increased in the presence of permeant divalent cations (15,
19). In
7-5-HT3 chimeric receptors,
the E172Q mutation abolishes the Ca2+-dependent
increase in potency (19).
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Ca2+,
Ba2+, and
Sr2+ carry inward currents through
E172Q/L247T nAChRs.
To determine whether the loss of modulation by permeant divalent
cations was due to the elimination of divalent permeation, we measured
the currents carried by Ca2+, Ba2+, and
Sr2+ in NMG-MeS solutions (Fig.
3). In an NMG-MeS bathing solution, ACh
evoked a small outward current at 60 mV that was probably carried by
intracellular K+, indicating that NMG+ does not
carry any inward current (Fig. 3, top trace). In NMG-MeS solutions containing 10 mM Ca2+, Ba2+, or
Sr2+, ACh evoked clear inward currents. The activation and
desensitization kinetics of ACh-evoked currents obtained in
Ca2+-, Ba2+-, or Sr2+-containing
NMG-MeS solutions were indistinguishable from those recorded in the
normal Na+-containing extracellular solution. Thus the loss
of Ca2+-, Ba2+-, and Sr2+-dependent
changes in ACh EC50 in E172Q
7
receptors was not due to an inability of these ions to permeate the
receptors. Quantitatively, the flux of divalent ions through E172Q/L247T
7 nAChRs was
significantly smaller than that of L247T
7
receptors (15). The currents of
E172Q/L247T
7 nAChRs carried by
10 mM Ca2+, Ba2+, or Sr2+ were
~10% as large as those carried by 96 mM Na+ (Fig. 3),
whereas the currents of L247T
7 receptors
carried by the divalent cations were the same size as those carried by
Na+ (15). These data suggest a role for
E172 in divalent ion permeation.
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Mutation of E172 reduces basal activity of
L247T 7 nAChRs.
In addition to having an extremely low EC50 for ACh,
L247T
7 receptors have a detectable level of
activity in the absence of agonist (5). This activity is
evident as an inhibition of basal current by MLA (32), a
potent and selective blocker of
7 neuronal nAChRs
(32, 44), including L247T
7
receptors. Permeant divalent cations cause a 10-fold increase in the
basal activity of L247T
7 receptors
(15). To determine whether this agonist-independent activity of L247T
7 receptors was altered by
mutations of E172, we measured the MLA-inhibited basal
currents of oocytes expressing E172Q/L247T
7 receptors.
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Mutation of E172 eliminates activation of
L247T 7 nAChRs by DH
E.
L247T
7 nAChRs are activated by DH
E, an
antagonist of wild-type neuronal nAChRs (3). In the
presence of permeant divalent cations, DH
E is a full agonist of
L247T
7 receptors (3, 15). To
determine whether E172 is critical for the activation of
L247T
7 receptors by antagonists, we tested
the efficacy of DH
E on E172Q/L247T
7 nAChRs. Figure 5,
left, confirms that activation and desensitization of ACh-
and DH
E-evoked responses of L247T
7
nAChRs were virtually identical. In contrast, DH
E failed to activate
E172Q/L247T
7 receptors (Fig. 5,
right), even at concentrations as high as 300 µM. In
ES-Ca2+, the maximal DH
E-evoked responses of
E172Q/L247T
7 receptors were
<0.04% of ACh-evoked responses (n = 9). In ES-EGTA,
no responses to DH
E were recorded (n = 4). In the
presence of an EC50 dose of ACh in ES-Ca2+ (38 µM), DH
E was an inhibitor of E172Q/L247T
7 nAChRs with an IC50 of ~1 µM (not
shown). This IC50 is similar to that recorded for wild-type
7 nAChRs (IC50 ~0.7 µM; see Ref. 9). DH
E also failed to activate
E172Q/L247T
7 nAChRs in the
presence of 2.5 mM Ba2+ or Sr2+ (n = 9).
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E172 is accessible to the aqueous milieu.
A model of the extracellular domain of 7 receptors,
based on the crystal structure of AChBP, suggests that E172
is at a position near the bottom of the outer vestibule of nAChRs (Fig.
6). E172 is located at the
interface between two adjacent subunits, and the side chain appears to
extend toward the lumen of the vestibule. Thus E172 may be
positioned to interact directly with permeant ions, congruent with the
observations that E172 is important for mediating the
increase in agonist potency of
7 receptors by
Ca2+ and other permeant divalent cations.
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DISCUSSION |
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We have shown that the E172Q and E172C
mutations of L247T 7 nicotinic receptors
eliminate receptor stimulation by the presence of permeable divalent
cations. ACh dose responses of E172Q/L247T
7 nAChRs were inhibited by the presence of
Ca2+, Ba2+, or Sr2+, and
E172C/L247T
7 nAChRs were
insensitive to the presence of these ions.
E172Q/L247T
7 nAChRs did not
display the high level of basal activity that is a characteristic of
L247T
7 receptors and were not activated by
the antagonist DH
E. E172C/L247T
7 receptors were blocked by the thiol-modifying reagent
MTSET, indicating that E172 is accessible to permeant ions.
These data support the conclusion drawn from a chimera of
7 nicotinic receptors and 5-HT3 receptors that E172 is essential for the modulation of
7 receptors by Ca2+ and other permeant
divalent cations (19). The data are also consistent with a
model of
7 receptors based on the crystal structure of
AChBP (6) that places E172 near the inner
surface of the vestibule, where it can come in contact with the
extracellular solution.
Our results do not support a kinetic model for the gating of
L247T 7 receptors in which the receptors
enter a conducting desensitized state (3). This model is
based on the observations that slowly desensitizing L247T
7 receptors are activated by agonists at very low
concentrations (suggesting binding to the high-affinity desensitized
states), are activated by antagonists, and have a high level of
agonist-independent basal activity (attributed to resting
desensitization). However, E172Q/L247T
7 nAChRs have a slow desensitization rate similar to
that of L247T
7 receptors, but
E172Q/L247T
7 nAChRs are not
activated by very low concentrations of ACh, are not activated by
DH
E, and do not have a high level of resting activity. Thus in
E172Q/L247T
7 nAChRs the
phenomenon of slow desensitization is not accompanied by other
characteristics suggesting that a desensitized state is conducting.
The data are consistent with an alternative model in which
L247T 7 receptors have altered activation
and desensitization kinetics (15, 16, 25). In this model,
the prolonged response of the receptor to agonists arises from a
decrease in the rate of entry into the desensitized state. Activation
by low concentrations of agonists, agonist activity of antagonists, and
elevated resting activity all derive from a dramatically increased
opening rate, increasing the intrinsic efficacy of the receptor
(10). In this interpretation, the
E172Q/L247T
7 receptors have a
slow desensitization rate similar to that of L247T
7 receptors but have an opening rate that is closer to
that of wild-type
7 nAChRs. We conclude that
L247T
7 nAChRs and other mutants derived
from L247T
7 receptors are a reasonable
model for wild-type
7 receptors because the mutations
alter the rates of transitions between closed, open, and desensitized
states but do not critically alter the nature of those states.
The observation that E172C/L247T
7 receptors were inhibited by extracellular MTSET is
consistent with a structural model of the vestibule of
7
nAChRs based on the crystal structure of AChBP (6). In the
model, E172 in chick
7 nAChRs
(R170 of AChBP) is located at the NH2 terminus
of the
9 strand, extending toward the central pore, where it could
contact the aqueous environment in the lumen of the pentamer (Fig. 6;
Ref. 6). The accessibility of cysteine at position 172 to
extracellular MTSET supports this vestibule-lining location of
E172. This glutamate is conserved in ACh receptors and
5-HT3 receptors, but not in AChBP, GABA receptors, or
glycine receptors (6), further supporting the view that it
may have a role in cation permeation and/or modulation. The
modification of the cysteine at position 172 by MMTS may inhibit the
receptor by altering interactions at the subunit-subunit interface. In
addition, it is possible that modification by MTSET and MMTS also
partially interferes with ion permeation.
Ca2+ interacts with one or more low-affinity binding sites
during permeation through 7 receptors (28).
Even though E172Q/L247T
7
receptors conduct divalent cations, they display divalent current
amplitudes that are smaller than those of L247T
7 receptors (15). Thus E172 may
be part of Ca2+-binding sites that are important in both
ion permeation and modulation. Additional binding sites that do not
involve E172 are also likely. Our results support the view
that the outer vestibule of nicotinic receptors, especially near the
junction between the vestibule and the transmembrane pore, plays an
essential role in the modulation of
7 receptors by
Ca2+ and other permeant divalent cations.
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ACKNOWLEDGEMENTS |
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We thank M. Ballivet (University of Geneva) for the
7 nAChR cDNA and C. Labarca and H. A. Lester
(California Institute of Technology) for the pAMV vector. We also thank
B. Temple (University of North Carolina at Chapel Hill) for assistance
with molecular modeling.
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FOOTNOTES |
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This work was supported by National Institute of Neurological Disorders and Stroke Grant NS-37317.
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.
July 17, 2002;10.1152/ajpcell.00204.2002
Received 3 May 2002; accepted in final form 16 July 2002.
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