0022-3565/98/2862-0619$03.00/0
THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS
Copyright © 1998 by The American Society for Pharmacology and Experimental Therapeutics
JPET 286:619 –626, 1998
Vol. 286, No. 2
Printed in U.S.A.
Two Domains of the Beta Subunit of Neuronal Nicotinic
Acetylcholine Receptors Contribute to the Affinity of
Substance P1
GRACE A. STAFFORD,2 ROBERT E. OSWALD, ANTONIO FIGL,3 BRUCE N. COHEN,3 and GREGORY A. WEILAND
Department of Pharmacology, College of Veterinary Medicine, Cornell University, Ithaca, New York (G.A.S., R.E.O., G.A.W.) and Division of
Biology, California Institute of Technology, Pasadena, California (A.F., B.N.C.)
Accepted for publication April 1, 1998
This paper is available online at http://www.jpet.org
The tachykinin SP is a neurotransmitter and neuromodulator in the central and peripheral nervous systems (Nicoll et
al., 1980). As a neurotransmitter, SP acts via the NK-1, a
member of the seven transmembrane, G protein-coupled receptor superfamily. The binding of SP to NK-1 receptors
leads to activation of phospholipase C, resulting in increased
inositol trisphosphate levels and the release of calcium from
intracellular stores (Mau and Saermark, 1991). In Xenopus
oocytes expressing cloned NK-1 receptors, the release of calcium causes the activation of chloride currents (Fong et al.,
1992). As a neuromodulator, SP has been shown to inhibit
agonist-induced nAChR activation, as Steinacker and Highstein (1976) first demonstrated at the Mauther fiber-giant
fiber synapse in the hatchet fish. Since then, SP has been
Received for publication October 28, 1997.
1
This work was supported by Grant BNS-8911782 from the National Science Foundation and by Cooperative State Research, Education, and Extension Service, USDA (Project Number NYC-425– 432) to G.A.W. and by Grant
RO1 NS 18660 from the National Institutes of Health to R.E.O. G.A.S. was
supported by the Cornell Biotechnology Institute and the Pharmaceutical
Manufacturers Association Foundation.
2
Current address: Wadsworth Center, Albany, NY 12201-2002.
3
Current address: Division of Biomedical Sciences, University of California
at Riverside, Riverside, CA 9521– 0121.
tors to the apparent affinity of substance P. The affinity of
acetylcholine was only affected by residue changes between
105 and 109. Site-directed mutagenesis revealed two amino
acids that are important determinants of the affinity of substance P, b4(V108)/b2(F106), which is in the middle of the first
extracellular domain, and b4(F255)/b2(V253), which is within
the putative channel lining transmembrane domain M2. However, other residues within these domains must be making
subtle but significant contributions, since simultaneous mutation of both these amino acids did not cause complete interconversion of the b subunit-dependent differences in the receptor affinity for substance P.
shown to modulate nicotinic responses of both neuronal
(Livett et al., 1979; Akasu et al., 1983; Clapham and Neher,
1984; Simasko et al., 1985; Simmons et al., 1990; Stafford et
al., 1994) and skeletal muscle (Akasu et al., 1983; Simasko et
al., 1985; Min and Weiland, 1992) nAChRs. These studies
have shown that noncompetitive inhibition by SP is a general
characteristic of nAChRs, most consistent with a direct interaction with the receptor at a unique site. This site has a
pharmacology distinct from that of the G protein-coupled NK
receptors. The evidence for a physiological role for this direct
modulation is strongest in the adrenal gland where SP-containing neurons innervate the chromaffin cells and SP modulates nAChR-mediated catecholamine secretion (Livett and
Zhou, 1991). SP may protect the nAChR from agonist-mediated irreversible deactivation (Boyd and Leeman, 1987) and
could be involved in maintaining catecholamine secretion
during stress (Livett and Zhou, 1991).
Muscle and neuronal nAChRs are pentameric proteins
forming ligand-gated ion channels (Changeux, 1990) that
mediate signal transmission at the neuromuscular junction
and in the central and peripheral nervous systems. Whereas
muscle receptors require four different subunits (a, b, g, and
ABBREVIATIONS: ACh, acetylcholine; DHbE, dihydro-b-erythroidine; G protein, heterotrimeric GTP binding protein; nAChR, nicotinic acetylcholine receptor; nBGT, neuronal bungarotoxin; NK, neurokinin; nH, Hill coefficient; SP, substance P; TMA, tetramethylammonium.
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ABSTRACT
Substance P is known to noncompetitively inhibit activation of
muscle and neuronal nicotinic acetylcholine receptors. Neuronal nicotinic receptors formed from different combinations of a
and b subunits exhibited differential sensitivity to substance P,
with those containing b-4 subunits having a 25-fold higher
affinity than those having b-2 subunits. To identify the regions
and/or amino acid residues of the b subunit responsible for this
difference, chimeric b subunits were coexpressed with a-3 in
Xenopus oocytes and the IC50 values for substance P were
determined. Amino acid residues between 105 and 109 (b4
numbering), in the middle of the N-terminal domain, and between 214 and 301, between the extracellular side of M1 and
the intracellular side of M3, were identified as major contribu-
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Stafford et al.
Materials and Methods
Construct and plasmid preparation. Plasmid containing
cDNA coding for rat a-3 was kindly supplied by Dr. Roger Papke
(University of Florida, Gainesville, FL). Chimeras between rat neuronal nAChR subunits b-2 and b-4 were generated as previously
described (Figl et al., 1992). The cDNA coding for some of the chi-
meric subunits were subcloned into the oocyte DNA expression vector pOEV (a gift of Dr. William L. Taylor, Vanderbilt University) at
the polylinker sequence which is located between a TFIIIA promoter
and an SV40 transcription terminator (Pfaff et al., 1990). Plasmids
were propagated in the Escherichia coli host (Dh5a strain) and
purified using the Wizard miniprep kit (Promega, Madison, WI).
mRNA was transcribed using SP6 and T3 Ampliscribe (Epicentre,
Madison, WI) and capped by the inclusion of diguanosine triphosphate.
Point mutations were introduced by sequential PCR (Cormack,
1994), using GeneAmp (Perkin Elmer Cetus, Norwalk, CT). For each
mutation, two primers were designed to match sites that were about
300 to 800 nucleotides apart and flanked the mutation site. Two
overlapping but oppositely oriented primers were designed to introduce the desired mutations. A silent restriction site was simultaneously introduced to allow screening using restriction enzymes. The
PCR-generated cDNA containing the mutations was digested with
restriction enzymes to cut two unique sites. The fragment was then
ligated into the original cloned gene that had also been cleaved with
the same enzymes. The vector was reintroduced into bacteria for
plasmid amplification and the inserts with the mutations were sequenced in the laboratory by the dideoxy method or at the Cornell
Biotechnology Program Sequencing Facility.
The nomenclature for the chimeras is that of Figl et al. (1992) and
identifies the residues from each subunit. The subunit from which
the amino terminus is derived is named first, with the ordinal denoting the number of N-terminal residues from that subunit. The
name of the subunit providing the remaining residues follows the
colon. Thus the chimera b-4 (105):b-2 contains the N-terminal 105
amino acids from b-4 and the remaining C-terminal residues from
b-2. The mutated residues are identified by their number in the
parent subunit, with the wild-type amino acid written first, followed
by the residue number, and then the amino acid to which it has been
changed, e.g., b-4(F255V).
Preparation and injection of Xenopus oocytes. Oocytes were
harvested from adult Xenopus laevis (Nasco, Fort Atkinson, WI)
under anesthesia (0.15% MS222) and manually dissected into groups
of several dozen. The follicle layers were removed by incubation in
Ca11-free oocyte saline solution (82.5 mM NaCl, 2.5 mM KCl, 1 mM
Na2HPO4, 15 mM HEPES, 1 mM MgCl2, pH 7.4) containing collagenase type I (1–2 mg/ml). Oocytes were maintained at 18°C in oocyte
saline solution (with 1 mM CaCl2) containing 5% horse serum, 5
U/ml penicillin, 5 mg/ml streptomycin and 150 mg/ml amikacin, and
the medium was changed daily. Four to five days before recording, 10
nl of DNA (;2 ng of plasmid DNA) was injected into the nucleus.
Alternatively, 50 nl of RNA (2 to 5 ng/subunit) was injected into the
cytoplasm 3 to 5 days before recording. Injections were made using
the Nanoject positive displacement oocyte injector (Drummond,
Broomall, PA).
Voltage-clamp measurements and analysis. Two electrode
voltage-clamp measurements were made at room temperature using
a Turbo Tec 01C amplifier (Adams & List, Westbury, NY). The
voltage electrode was filled with 3 M KCl and had a resistance of 0.4
to 2 MV. The current electrode was filled with 250 mM CsCl, 250 mM
CsF and 100 mM EGTA, pH 7.3. The resistance of the current
electrode was between 0.5 and 2 MV. Cells were routinely voltageclamped at -70 mV. Bath solution (oocyte saline with 1 mM CaCl2
and 1 mM atropine to prevent activation of muscarinic acetylcholine
receptors) was delivered at ;6 ml/min through a linear perfusion
system to oocytes placed in a Delrin chamber with a total volume of
0.45 ml. ACh/peptide solutions were delivered by preloading 2 ml in
a loop at the terminus of the perfusion system using a syringe. A
Mariotte flask filled with oocyte saline solution was used to maintain
constant hydrostatic pressure, and the ACh/peptide application was
initiated by a computer-triggered stream-switching valve (Rainin,
Emeryville, CA). The time between applications was 6 to 10 min to
allow recovery from ACh-induced desensitization. Data were collected on-line with an IBM AT computer using software developed in
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d), functional neuronal receptors can be formed from a combination of a and b subunits (Boulter et al., 1987) or, in
certain cases, a single type of a subunit (Couturier et al.,
1990). The family of neuronal nAChR subunits continues to
grow and there are currently eight a and three b neuronal
receptor subunits (Papke, 1993; McGehee and Role, 1995).
Both types of subunits of neuronal receptors have been
shown to be involved in determining the sensitivity of the
receptor to agonists and antagonists (Luetje and Patrick,
1991; Figl et al., 1992; Papke et al., 1993; Harvey and Luetje,
1996). Because it has been demonstrated that the g and d
subunits of the muscle nAChR play a role in agonist and
antagonist binding (Sine and Claudio, 1991; Czajkowski et
al., 1993; Sine, 1993), the involvement of both neuronal subunits is not surprising. Moreover, given the heterogeneity of
neuronal nAChR responses in vivo, the “mix and match” of
various subunits probably provides the molecular basis for
diversity of function (Papke, 1993).
Some of the structural determinants for SP modulation of
nAChRs are now becoming apparent. Min et al. (1993) found
that the g and d subunits of Torpedo nAChRs were affinity
labeled with either [3H]SP and a bifunctional cross-linker or
the photoaffinity reagent [125I]p-benzoylphenylalanine-SP.
Blanton et al. (1994) demonstrated that [125I]p-benzoylphenylalanine-SP labeled the M2 region of the Torpedo d subunit. Using the oocyte expression system we recently found
that the b subunit of the neuronal receptor contributes to the
IC50 for SP inhibition, with b-4 subunit-containing receptors
having a 25-fold higher apparent affinity for SP than b-2containing receptors, whether coexpressed with a-3 or a-4
(Stafford et al., 1994). These findings suggested that, using
molecular biological approaches and the Xenopus oocyte expression system, the structural domains of the nAChR involved in the interaction of SP with the nAChR might be
resolved as they had been previously for several agonists and
antagonists (Figl et al., 1992; Luetje et al., 1993; Papke et al.,
1993).
We undertook to identify the region(s) and amino acid(s)
responsible for the difference in the IC50 for b-4- vs. b-2containing receptors, taking advantage of the significant sequence similarities between the subunits. A series of chimeric b-4/b-2 subunits were expressed with the a-3 subunit
in Xenopus oocytes and by quantitating the inhibition of
agonist-induced current by SP, we were able to identify two
regions that appeared to be the determinants of the difference between the subunits. Site-directed mutagenesis of the
candidate residues individually demonstrated the importance these amino acids; however, mutation of both residues
together was not sufficient to completely interconvert each
receptor’s sensitivity to SP. It is apparent from these results
that more global structural and conformational issues are
involved in the binding and inhibition of nAChR activity by
SP which cannot be duplicated with two single amino acid
changes.
Vol. 286
nAChR b Subunit Domains and Substance P
1998
the laboratory. Current traces were recorded at the same time on a
chart recorder. The digitized recordings were transferred from the
IBM AT to a Sun 4/330 computer for further analysis using PLOT
(Gradient Software, Ithaca, NY).
ACh activation curves and SP inhibition curves were analyzed by
nonlinear least squares fitting using KaleidaGraph (Synergy Software, Reading, PA) on a Macintosh computer. The EC50, apparent
Hill coefficient (nH), and maximum current (Imax) for ACh were
estimated from the concentration dependence of ACh-induced current using a form of the Hill equation:
I5
I max@ ACh#
@ ACh#
nH
nH
n
1 EC 50H
(1)
I5
I maxIC 50
@ SP# 1 IC 50
(2)
where I is the peak current in the presence of [SP] and Imax is the
peak current in the absence of SP. When a Hill coefficient was
incorporated into equation 2, the values of nH determined from the
fits were not significantly different from 1. To compare SP sensitivity
at comparable current responses, SP dose-response curves were determined at ACh concentrations near the EC50 value for the subunit
combination tested.
Results
Dependence of inhibition by substance P on subunit
structure. Because of the significant sequence identity between the beta subunits (63% overall and more than 90% in
some regions, see fig. 1), a series of chimeric b-4/b-2 constructs were coexpressed with a-3 in Xenopus oocytes to
attempt to identify the regions responsible for the 25-fold
difference in the IC50 of SP for b-4- vs. b-2-containing receptors (fig. 2). The chimeric beta subunits examined focused on
two potentially important areas, the extracellular N-terminal domain [b-4(214):b-2, b-4(116):b-2, b-4(113):b-2,
b-4(111):b-2, b-4(109):b-2 and b-4(105):b-2] and the first
three transmembrane domains [b-4(301):b-2, b-4(214):b-2
and b-2(299):b-4] which include the putative pore-lining M2
region. We expected the N-terminal region to be important
since it comprises more than 90% of the extracellular domain
and had previously been shown to be important in the interaction of agonists with the receptor (Figl et al., 1992). We
were also particularly interested in the M2 region, because
biochemical studies had indicated it contributed to the binding site of SP (Min et al., 1993; Blanton et al., 1994). The large
intracellular loop and fourth transmembrane region were
also examined [b-4(301):b-2, b-4(325):b-2, b-2(299):b-4,
b-2(323):b-4 and b-2(421):b-4]. Based on the results of these
studies, several point-mutated subunits were generated and
characterized (fig. 2).
SP inhibited ACh-induced currents for all a3b subunit
combinations that expressed functional receptors. Representative current traces for the effect of 5 mM SP on AChinduced current for several subunit combinations are shown
in figure 3. Because the EC50 for ACh was dependent on the
subunit combination expressed, in order to be able to compare IC50 values for SP, the concentration-dependences of SP
inhibition were determined for each subunit combination at
an ACh concentration within less than a factor of 2 of its
EC50 value (fig. 2). Determination of the EC50 values for
activation by ACh for each subunit combination is presented
in the next section.
From the concentration-dependences of SP inhibition for
the chimeras, it was apparent that both the N-terminal extracellular domain and the region of the first three transmembrane domains contributed to the difference in the af-
Fig. 1. Aligned amino acid sequences of b2 (top)
and b4 (bottom). Dots between sequences indicate identical residues. The four putative transmembrane regions are denoted by horizontal
bars.
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where I is the peak current measured in the presence of [ACh].
Because of the slow perfusion system used, the current profiles
reflect the time-dependent sum of the kinetics of drug diffusion,
channel activation and desensitization, which in general will increase the EC50 value. Additionally, at high agonist concentrations,
channel blockade by ACh is likely (Sine and Steinbach, 1984). The
IC50 for SP was determined using the equation:
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Stafford et al.
Vol. 286
Fig. 3. Currents induced by application of acetylcholine (solid line) or
acetylcholine plus 5 mM substance P (broken line). Drugs were applied for
20 sec, as shown by horizontal bar. Current traces shown in the presence
and absence of SP are from the same oocyte. The concentrations of ACh
used were 10 mM for a3b2(F106V;V253F), 25 mM for a3b4(109):b2 and
a3b2(323):b4 and 100 mM for the others.
finity for SP (figs. 2, 4 and 5). About half of the difference
between b4 and b2 resided between b4(105) and b4(109) and
half between b4(214) and b4(301).
The receptor containing the first 105 b4 amino terminal
residues exhibited an IC50 for SP such as a3b2 (61 vs. 67 mM,
fig. 2). Extending the number of b4 residues by only four
more amino acids to 109 shifted the SP inhibition curve to the
left, about halfway to the value for a3b4 (14 vs. 3.3 mM; figs.
2, 4 and 5). Additional b4 residues to 214 resulted in no
further decrease in the IC50. Thus the sequence between 105
and 109 appeared to contain the amino acid residue(s) important for at least half of the difference in SP sensitivity
between b4 and b2. Comparison of the amino acid sequences
of the two subunits in this region revealed only a single
amino acid difference (fig. 1). At position 108, b4 has a valine,
while b2 has a phenylalanine at the homologous position
(106).
Based on these results with the chimeras, it was expected
that substitution of a phenylalanine for the valine in b4 at
108 and a valine for the phenylalanine in b2 at 106 would
result in receptors exhibiting SP sensitivity about halfway
between the two wild-types. Although this was partially true
for b4(V108F), which was approximately 3-fold less sensitive
to SP than wild-type b4 (IC50 5 9.8 vs. 3.3 mM; figs. 2 and 6),
b2(F106V) resulted in receptors that had a slightly lower
affinity for SP than a3b2 wild-type (IC50 5 148 vs. 67 mM; fig.
2).
Approximately half of the affinity difference between the b
subunits appeared to lie between b4(214) and b4(301), which
encompasses the transmembrane domains M1-M3. Within
this region there are only five amino acid residues that differ
between b4 and b2 (fig. 1). Of these, only one is a nonconservative difference, b4(F255)/b2(V253). Significantly, this residue is within M2, the putative lining of the ion channel pore.
In addition to the evidence that the M2 region was involved
in the binding of SP (Blanton et al., 1994), residue 255/253
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Fig. 2. Linear maps and IC50 values for substance P inhibition and EC50 values for acetylcholine-induced currents of chimeric and mutated b subunits
coexpressed with a3. Black areas represent amino acids from b4, white areas represent amino acids from b2, amino acid residues are numbered at
the bottom, and lines above the subunits denote putative transmembrane regions. Inhibition by SP was determined at a concentration of ACh within
a factor of 1.7 (0.60 –1.43) of its EC50 value and the responses for each oocyte were normalized to the current measured in the presence of ACh alone.
IC50 values and standard errors were obtained by nonlinear fitting equation 2 to all the data from each subunit/construct (6 –24 points). To determine
EC50 values for ACh, peak current was measured in the presence of increasing concentrations of ACh and normalized to 100 mM ACh. The parameters
of ACh activation were determined by fitting equation 1 to all the data from each subunit/construct. Hill coefficients ranged from 0.71 6 0.08 (b2) to
2.5 6 0.09 (b4(105):b2). Data from each subunit combination were pooled from (n) oocytes. The constructs b2(107):b4, b2(109):b4, b2(111):b4,
b4(423):b2 and b2(V253F) did not produce measurable ACh-induced currents when coinjected with a3, presumably for technical rather than structural
reasons. ND, Not determined.
1998
nAChR b Subunit Domains and Substance P
623
Fig. 4. Concentration-dependence of substance P inhibition of acetylcholine-induced currents for wild-type b4
and b2 and several chimeric b4:b2 subunits coexpressed
with a3. Peak current responses to ACh were measured
in the presence of increasing concentrations of SP. ACh
concentrations were 100 mM for a3b4, a3b4(301):b2,
a3b4(214):b2, a3b4(116):b2, and a3b4(105):b2; 50 mM
for a3b2. Data for each subunit combination are from
three to six oocytes. For each oocyte the responses were
normalized to the current measured in the presence of
ACh alone. Each point is the mean of one to four measurements at each concentration. Error bars represent
the S.E.M.; if there are no error bars, SEM is smaller
than the symbol or only one determination was made at
that concentration. The lines are nonlinear fits of equation 2 to all the data from each subunit/construct (6 –24
points).
was chosen for investigation because mutations in the pore
region have been shown to affect the interaction of channel
blockers (Leonard et al., 1988; Charnet et al., 1990) and to
alter the pharmacological properties (Bertrand et al., 1992;
Devillers-Thiéry et al., 1992) of the receptor. Receptors containing valine substituted for phenylalanine in b4
[a3b4(V255F)] had an IC50 value for SP (19 mM), increased
about halfway to that of the a3b2 wild-type (fig. 2), consistent
with a significant contribution of this residue either to the
binding site of the peptide or to the transduction of binding
into inhibition of receptor activation. Unfortunately,
a3b2(V253F) did not produce functional receptors, despite
several attempts.
Because the single mutation at b4(F255V) reduced the
sensitivity of the receptor to SP about halfway to a3b2 and
the point mutation b4(V108F) had also reduced the sensitivity towards wild-type b2, these residues appeared to be critical determinants of the difference in SP affinity for the b
subunits. If the structural differences created by these residue changes were independent, then the double point mutation of b4 (V108F; F255V) should result in the conversion of
b4 affinity for SP into that of b2. As shown in figures 2 and
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Fig. 5. IC50 values for SP and EC50 values for ACh of receptors containing a3 and chimeric b subunits. Value of zero amino acids on the x-axis is
the b2 subunit, 475 is b4. Data are from figure 2. Lines were drawn to
connect data points which appear continuous. Discontinuities in the IC50
values between b4 residues 105 and 116 and between 214 and 301 reflect
the importance of these regions in the interaction of SP with the receptor.
6, this was not the case and the double mutant had approximately the same affinity for SP as either single mutation
had, about halfway between b2 and b4. This was also found
to be true for the double mutation of b2 (F106V/V253F),
which displayed an affinity for SP about halfway between the
wild-type b subunits (fig. 2).
Based on these results, we hypothesized that although the
residues preceding 108/106 initially did not seem to be important for the difference in sensitivity, they might be indirectly involved and could be important in determining the
three dimensional structure around the amino acid at
b4(108)/b2(106). To investigate this, a point mutation at
b2(V253F) was introduced into the chimeric subunit
b4(109):b2 to generate b4(109):b2(V253F), a b subunit with
the first 109 amino acids from b4 and the remainder from b2,
except that the M2 domain is b4. Although receptors formed
with this b construct were somewhat more sensitive to SP
than those containing the two point mutations b2(F106V;
V253F) (IC50 5 14 vs. 32 mM; fig. 2), the apparent affinity
was not significantly different from that of the chimera
b4(109):b2.
Acetylcholine dose-responses. The EC50 value of ACh
for each subunit/chimera/mutation combined with a3 was
determined so that SP inhibition could be investigated using
concentrations of ACh that gave comparable relative responses. A 20-sec application of ACh induced activation of
inward cationic currents, as shown in figure 3 (solid lines).
EC50 values for all the expressed receptor subunit combinations are shown in figure 2. In this study we observed only
about a 4-fold difference in EC50 values of ACh for receptors
containing b2 or b4 subunits (37 vs. 143 mM, fig. 2). This is in
contrast to the near 20-fold difference (10 vs. 210 mM) previously reported (Cohen et al., 1995). The discrepancies in the
EC50 values for ACh most likely reflect 1) differences in the
method of agonist application (in the current study we used
a relatively slow bath perfusion, while the previous study
used a more rapid U-tube application), 2) differences in the
holding potential (-70 mV in the current study and -50 mV in
the previous report) and 3) differences in the composition of
perfusion solutions, most notably 1 mM atropine was included in the current study. It is not unexpected that differences in the rate of drug application would cause discrepan-
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Stafford et al.
Vol. 286
Fig. 6. Concentration-dependence of substance P inhibition of acetylcholine-induced currents for wild-type
b4 and b2 and point-mutations of b4 coexpressed with
a3. ACh concentrations were near the EC50 value determined for each mutant as shown in figure 2. Data for
each subunit combination are from two to five oocytes.
For each oocyte the responses were normalized to the
current measured in the presence of ACh alone. Each
point is the mean of one to six measurements at each
concentration. Error bars represent the S.E.M.; if there
are no error bars, S.E.M. is smaller than the symbol or
only one determination was made at that concentration. The lines are nonlinear fits of equation 2 to all the
data from each subunit/construct (6 –24 points).
tations had little effect of the affinity for ACh, although
b4(V108F) was not significantly different from b2 and
b2(F106V;V253F) had an unexpectedly high affinity for agonist.
Discussion
We had previously found that neuronal nAChRs containing b4 subunits have a higher affinity for SP than do b2containing receptors, whether they are coexpressed with a3
or a4 (Stafford et al., 1994). We identified two separate regions of the b subunit that are important for this difference in
the sensitivity of the receptors to SP: between b4(105) and
b4(116) and between b4(214) and b4(301). Chimeric subunits
containing portions of the two b subunits were used to locate
these regions, and point mutations were introduced to attempt to determine the individual amino acids involved. The
amino acids V108 and F255 of b4 and the homologous b2
residues (F106 and V255) were identified as important determinants of the affinity for SP, although the double mutations did not convert one subtype to the other, indicating
other residues are making significant, but more subtle, contributions to the interaction with the peptide.
The b subunit has previously been shown to affect many
properties of neuronal nAChRs, including the single channel
characteristics. Papke and Heinemann (1991) have shown
that the b subunit affects the rate of ACh dissociation and the
rate of channel opening. Receptors containing b4 are much
more sensitive to the ganglionic stimulants cytisine and nicotine (Luetje and Patrick, 1991; Figl et al., 1992) but much
less sensitive to the neurotransmitter ACh (Cohen et al.,
1995) and the antagonists DHbE (Harvey and Luetje, 1996)
and nBGT (Papke et al., 1993; Harvey and Luetje, 1996) than
are b2 containing receptors. Chimeric b4/b2 subunits have
been used to map the regions responsible for most of these
differences. From these studies it is clear that the extracellular N-terminus is the most important region of the b subunit for the interactions of these compounds with the receptor. Amino acids b4(108) and b4(110) can account for much of
the relative sensitivity to cytisine, although the difference in
nicotine sensitivity could not be localized to a particular
region of the b subunit (Figl et al., 1992). The first 121 amino
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cies in the quantitative values determined (especially EC50
values of agonists where desensitization can significantly
affect the peak current observed). For example, Harvey and
Luetje (1996) reported only a 3-fold difference in EC50 values
of ACh for a3b2 vs. a3b4 receptors (71 vs. 210 mM), using a
relatively slow perfusion method and with a holding potential of 270 mV. It should be noted that despite these differences in EC50 values, both these studies and the current one
(data not shown) found significant differences in the apparent cooperativity for ACh activation of b2- vs. b4-containing
receptors. For all these studies the Hill coefficient for ACh
was near 1.0 for a3b2 and near 2.0 for a3b4.
The b4:b2 chimeras that contained more than the first 116
N-terminal residues of b4 exhibited a high EC50 value (a3b4like), as did a3b4(105):b2 (figs. 2 and 6). The three chimeras
between b4(105) and b4(116), however, displayed low EC50
values, such as b2. This region (b4(105–116)/b2(103–114))
had previously been shown to be a structural “hot spot” for
the action of the partial agonists cytisine, TMA and nicotine
(Figl et al., 1992) and acetylcholine (Cohen et al., 1995), and
may contribute to an agonist binding site that bridges the a
and b subunits in neuronal receptors (Cohen et al., 1995).
With the exception of b4(V108F), the single point mutations had little effect on the EC50 values of the receptors (fig.
2). The EC50 value for a3b2(F106V) was essentially that of
a3b2 and a3b4(F255V) was not significantly different from
a3b4. However, a3b4(V108F) had an EC50 value no different
from that of a3b2. Combining the two mutations in b4 resulted in a receptor, a3b4(V108F;F255V), with an EC50 (110
mM) close to wild-type a3b4. a3b2(F106V;V253F) was the
most sensitive of all the receptors to ACh (EC50 5 7 mM). The
chimera with a point mutation, b4(109):b2(V253F), had an
EC50 (78 mM) like wild-type b4, in contrast to its parent
chimera, b4(109):b2, which was more like b2.
Thus as previously reported (Cohen et al., 1995), the difference in the affinity for ACh appears in large part to be
determined by the first 116 N-terminal residues of the b
subunit, with the conversion from the high affinity b2 form to
the low affinity b4 form occurring between 108 and 116 of b4.
Unlike for substance P, however, the most critical residues
appear to be b4(115) and b4(116), where conversion from
high to low affinity for ACh occurred. In general point mu-
1998
625
not involved directly in the binding of SP. These residues
may contribute to the agonist binding site as suggested by
Cohen et al. (1995) or may participate in conformational
changes involved in agonist-induced activation or desensitization, which could indirectly affect the apparent affinity of
SP by altering agonist properties. Even if these residues are
within the agonist binding site, they can contribute to the
gating properties of the channel as has been shown, for
example, by Chen et al. (1995) who found mutation of tyrosine 190 within the ACh binding site of the a-subunit
affected both agonist binding and activation kinetics (for
review see Arias, 1997).
Between residues b4(214) and b4(301) there are five amino
acid differences, but only a single nonconservative change. In
the M2 region, b2 has a valine at 253 and b4 has a phenylalanine at the comparable position (255). The receptor
a3b4(F255V) had an IC50 for SP about halfway between the
two wild-type receptors. We believe that the single residue
b4(255) alone can account for the difference in SP sensitivity
mapped to between b4(214) and b4(301) because the difference in the IC50 between wild-type b4 and b4(F255V) (3.3 vs.
19 mM) is essentially the same as the difference between
b4(301):b2 and b4(214):b2 (5.5 vs. 20 mM). Because
a3b2(V253F) would not express, the effect of the single mutation in M2 of b2 remains unknown. The amino acid
b4(F255) [and the homologous residue, b2(V253)] is located
in the middle of the putative second transmembrane domain,
which is the believed to line the channel pore (Imoto et al.,
1986; Oiki et al., 1988). b4(F255)/b2(V253) is four amino
acids nearer the extracellular mouth of the receptor than the
highly conserved leucine [b2(L249)/b4(L251), see fig. 1] that
is thought to face the lumen and contribute to the narrowest
region of the pore (Unwin, 1993). Therefore, it should also
face the lumen of the pore, whether M2 is an a-helix or
b-structure. It is not unlikely that SP is binding in the channel near this residue, because Blanton et al. (1994) have
cross-linked the affinity label [125I]p-benzoylphenylalanineSP to the M2 region of the d subunit from Torpedo. The
greater sensitivity of b4 containing receptors to SP inhibition
could then be explained by the stabilization of the positive
charges on SP by the aromatic p electrons of the phenylalanine, much as the aromatic amino acids stabilize ACh in the
binding pocket of acetylcholinesterase (Dougherty and
Stauffer, 1990; Sussman et al., 1991). This could account for
the reduction in SP IC50 for a3b2(F106V;V253F) receptors
over a3b2(F106V) receptors. However, longer range effects
cannot be ruled out, as found with mutations in M2 of a7
where agonist and antagonist interactions with the receptor
were affected by point mutations in the channel region (Revah et al., 1991; Bertrand et al., 1992; Devillers-Thiéry et al.,
1992).
Combining the two single mutations in b4 did not produce
an additive effect, and did not result in the predicted conversion of b4 to b2 sensitivity to SP. a3b4(V108F;F255V) receptors had essentially the same IC50 as a3b4(F255V). The
double mutant in the b2 subunit, a3b2(F106V;V253F) had an
intermediate IC50 for SP, demonstrating that the phenylalanine in the M2 could render the b2 containing receptor more
sensitive to SP. However, changing b2(253) from a valine to
a phenylalanine in the chimera b4(109):b2 had no effect on
SP sensitivity. This suggests, as discussed previously, that
the differences in affinity for substance P are not due to only
Downloaded from jpet.aspetjournals.org at ASPET Journals on June 17, 2017
acids of the b subunit determined the kinetics of nBGT block
(Papke et al., 1993). The major determinant of DHbE and
nBGT affinity was shown to be b4(K61)/b2(T59) with other
minor determinants in the first 100 residues of the N-terminus (Harvey and Luetje, 1996). Chimeras of b4/b2 subunits
were used by Cohen et al. (1995) to demonstrate the importance of the first 120 residues in determining the EC50 for
ACh, with residues between b2(104) and (120) accounting for
the relative sensitivity of a3b2 to cytisine, TMA, and ACh.
Chimeric subunits followed by site-directed mutation have
been used to identify amino acids of the g and d subunits
involved in curare binding to muscle nAChRs (Sine, 1993)
and of the a subunits that contribute to agonist and antagonist sensitivity of neuronal nAChRs (Luetje et al., 1993). Sine
(1993) expressed g/d chimeras to identify two regions in
mouse g and d subunits that were determinants of curare
affinity. Interestingly, two of the residues identified in the
mouse g subunit (I116 and Y117) are homologous to b subunit residues b2(I118) and b2(F119) that are very near the
region we found to affect ACh and substance P interactions
with the receptor (see fig. 1). Luetje et al. (1993) used chimeric a2/a3 subunits expressed with b2 in Xenopus oocytes
to identify the determinants of nBGT sensitivity and the
relative nicotine/ACh sensitivities between a2 and a3 coexpressed with b2.
We have identified two regions of the b subunit that were
responsible for the difference in sensitivity to SP inhibition of
ACh-induced current in nAChRs. Receptors containing chimeric b subunits with the first 105 amino terminal residues
from b4 displayed the same sensitivity as b2-containing receptors. If the amino terminal b4 residues were extended to
109, the resultant receptors displayed SP sensitivity intermediate between a3b2 and a3b4. Extending the amino terminal b4 residues to 214, thus including the entire N-terminal extracellular domain, had no additional effect and
resulted in receptors with similar intermediate sensitivity.
However, when the chimeric b subunits contained b4 amino
terminal residues past the third transmembrane domain
(;300 amino terminal residues), the receptors displayed SP
sensitivity of wild-type a3b4. Similarly, receptors with chimeric subunits having b2 past M3 had wild-type a3b2 SP
sensitivity. Each of these regions, b4(105–109) and b4(214 –
301), accounted for approximately half of the difference in
sensitivity to SP of the two wild-type receptors (see figs. 2
and 5).
There is a single amino acid in the region of b4(105) and
b4(109) that differs between b2 and b4: a phenylalanine at
b2(106) is replaced by a valine at b4(108). It was expected
that substitution of that residue with the homologous one
would result in receptors with intermediate IC50 values for
SP. Receptors with the single mutation a3b4(V108F) were
only slightly less sensitive to SP than wild-type a3b4, and
a3b2(F106V) had an IC50 for SP of about 150 mM, much
greater than a3b2 wild-type. This lack of reciprocity when
exchanging residues between subtypes was also seen by
Luetje et al. (1993) for the relative sensitivity of nicotine vs.
ACh, and probably reflects subtle, but significant, contributions by other residues. Because this region has also been
shown to be important in the interaction of agonists (fig. 5;
Figl et al., 1992; Cohen et al., 1995) and inhibition by SP is
noncompetitive (Stallcup and Patrick, 1980; Simasko et al.,
1987; Stafford et al., 1994), it is most likely that this region is
nAChR b Subunit Domains and Substance P
626
Stafford et al.
these two amino acids, but other residues are involved, making subtle, three-dimensional structural contributions that
are not apparent from the results with the chimeras.
Using chimeric b subunits coexpressed with a3 in Xenopus
oocytes, it has been possible to define two areas of the b
subunit that contribute to the differences in sensitivity to SP
of b2- and b4-containing nAChRs. Although the two amino
acids identified could not account for all of the difference,
b4(108) and b4(255) [and the b2 homologs, b2(106) and
b2(253)], clearly play important roles in the inhibition of
nAChR activation by SP. Most likely b4(255) is involved in
the binding of SP to the receptor while b4(108) may be
involved in agonist binding and/or receptor activation and
indirectly participate in the inhibitory action of the peptide.
Acknowledgments
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Send reprint requests to: Dr. Gregory A. Weiland, Department of Pharmacology, College of Veterinary Medicine, Cornell University, Ithaca, NY 14853.
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The authors thank Drs. Jim Boulter, Roger Papke, Marc Ballivet
and William L. Taylor for supplying cDNA and plasmids; Dr. Roger
Papke for helpful discussions and Ms. Chris Bian for her excellent
technical assistance.
Vol. 286