|
|
|
|
Vol. 54, Issue 6, 1124-1131, December 1998
4
2 Nicotinic
Acetylcholine Receptor Expressed in Xenopus laevis
Oocytes
Research Institute of Toxicology, Utrecht University, Utrecht, The Netherlands
| |
Summary |
|---|
|
|
|---|
Nicotinic receptors generally are presumed to consist of two
and
three non-
subunits. We varied the relative levels of expression of
the neuronal nicotinic
4 and
2 receptor subunits in
Xenopus laevis oocytes by nuclear injection of cDNAs
coding for these subunits in
:
ratios of 9:1, 1:1, and 1:9. The
sensitivities of the receptors to acetylcholine and
d-tubocurarine were investigated in voltage-clamp
experiments. For receptors expressed at the 9:1 and 1:1
:
ratios,
the EC50 value of acetylcholine is ~60 µM. For the majority of the receptors expressed at the 1:9
:
ratio, the sensitivity to acetylcholine is enhanced 30-fold. No evidence for
more than one type of acetylcholine binding site in a single receptor
is obtained. The sensitivity to d-tubocurarine decreases with decreasing
:
ratio. IC50 values of
d-tubocurarine are 0.2, 0.5, and 2 µM for
the 9:1, 1:1, and 1:9
:
ratios, respectively. At the 1:9
:
ratio, additional receptors with an IC50 value of 163 µM d-tubocurarine are expressed. At least
two components with distinct sensitivities to
d-tubocurarine are required to account for the shift in
IC50. The combined agonist and antagonist effects reveal
four distinct subtypes of
4
2 nicotinic receptors. The results
imply that the subunit stoichiometry of heteromeric
4
2
acetylcholine receptors is not restricted to 2
:3
.
| |
Introduction |
|---|
|
|
|---|
Neuronal
nAChRs are ligand-gated ion channels present throughout the central and
peripheral nervous systems. Multiple neuronal nAChR subunits (
2-9
and
2-4) have been identified. These subunits combine in various
compositions to form pentameric receptors with different functional and
pharmacological characteristics (reviewed in McGehee and Role, 1995
).
The
4 and
2 nAChR subunits are expressed abundantly in brain
(Wada et al., 1989
), and the most common nAChR in the brain is composed of these two subunits (Whiting et al., 1990
;
Flores et al., 1992
). The subunit stoichiometry of
4
2
nAChRs expressed in Xenopus laevis oocytes has been deduced
to be
4:
2 = 2:3 (Anand et al., 1991
; Cooper
et al., 1991
), and the subunits are assumed to be arranged
around the central ion channel pore in the order 



(Anand
et al., 1991
). Patch-clamp recording from oocytes expressing
pairs of
and
nAChR subunits revealed ACh-gated single channels
with distinct conductances (Papke et al., 1989
; Papke and
Heinemann, 1991
; Charnet et al., 1992
; Kuryatov et
al., 1997
). The frequency of occurrence of the different
single-channel conductance states depends on the
:
ratio of the
mRNA mixture injected into the oocyte, indicating that receptors with
different subunit stoichiometry are expressed (Papke et al.,
1989
).
The agonist binding sites of heteropentameric neuronal nAChRs generally
are supposed to be located at the two 
subunit interfaces (reviewed in Bertrand and Changeux, 1995
). Functional nAChRs with distinct subunit stoichiometries might differ with respect to the
number and properties of agonist binding sites. We investigated the
functional properties of the agonist binding sites in X.
laevis oocytes expressing different levels of
4 and
2
nAChR subunits. The results on concentration-dependent effects of the
agonist ACh and of d-TC, a competitive antagonist of
4
2 nAChRs
(Bertrand et al., 1990
), demonstrate four distinct subtypes
of
4
2 nAChRs.
| |
Experimental Procedures |
|---|
|
|
|---|
Materials.
X. laevis were bred and
kept in the Hubrecht Laboratory (Utrecht, The Netherlands) or were
bought from Nasco (Fort Atkinson, WI) and kept in our own laboratory.
Neomycin was obtained from Sigma Chemical (St. Louis, MO). d-TC was
from Fluka (Buchs, Switzerland). All other materials used came from
sources identical to those described previously (Zwart and Vijverberg,
1997
).
Receptor expression.
Oocytes were prepared, injected, and
incubated in Barth's solution containing 5 mg/liter of neomycin at
19° for 3-7 days as described previously (Zwart et al.,
1995
; Zwart and Vijverberg, 1997
). Plasmids coding for
4 and
2
subunits of neuronal nAChRs were dissolved in distilled water at the
approximately equal concentrations of 152 ± 15 and 136 ± 12 µg/ml (mean ± standard deviation of triplicate spectrophotometric determinations), respectively. Unless noted otherwise, mixtures of these solutions at 1:9, 1:1, and 9:1 ratios were
coinjected at 18.4 nl/oocyte.
Electrophysiology and data analysis.
Oocytes were placed in
a silicon rubber tube (diameter, 3 mm), penetrated by two
microelectrodes, and voltage clamped at
80 mV. ACh and d-TC,
dissolved in external solution, were applied by perfusion of the tube
at a rate of ~20 ml/min. The external saline contained 115 mM NaCl, 2.5 mM KCl, 1 mM
CaCl2, and 10 mM HEPES, pH 7.2, with
NaOH. Voltage-clamp equipment, experimental protocols, and data
acquisition were exactly as described previously (Zwart and Vijverberg,
1997
). All amplitudes of ACh-induced ion currents were normalized to
the amplitude of alternately evoked control responses to a near-maximum
effective concentration of ACh to adjust for small variations in
response amplitude over time. Data are expressed as mean ± standard deviation of number of oocytes. ANOVA was performed using
Microsoft Excel. Results were compared using a two-tailed Student's
t test. Concentration-effect curves were fitted, using
Jandel SigmaPlot software, to data obtained in separate experiments,
and mean ± standard deviation values of estimated parameters were
calculated for three oocytes. Curves drawn in the figures were
generated using the mean values of the estimated parameters. Agonist
data were fitted according to the equations
i/imax = Emax/[1 + (EC50/[ACh])nH]
and i/imax = Emaxa/(1 + EC50a/[ACh]) + Emaxb/(1 + EC50b/[ACh]) for one- and two-component
activation curves, respectively. Antagonist data were fitted according
to the equations: i/imax = 1/[1 + ([dTC]/IC50)nH]
and i/imax = Em/[1 + ([dTC]/IC50a)nHa) + (1
Em)/[1 + ([dTC]/IC50b)nHb]
for one- and two-component inhibition curves, respectively. In these
equations, [ACh] and [dTC] are the concentrations of ACh and d-TC,
respectively; nH is the Hill
coefficient; and i/imax is the
normalized current amplitude. Goodness-of-fit was judged by analysis of
the residuals (run test).
| |
Results |
|---|
|
|
|---|
Effects of
4:
2 ratio on ion current amplitude.
Superfusion of voltage-clamped oocytes, expressing
4
2 nAChRs
after the injection of
4 and
2 subunit cDNAs in the
:
ratios 1:9, 1:1, and 9:1, with 300 µM ACh results in
ligand-gated ion currents. The effect of subunit ratio on response
amplitude was investigated in the same batch of oocytes because
expression levels vary between batches. The amplitude of the inward
current induced by the near-maximal effective concentration of 300 µM ACh at the holding potential of
80 mV depends on the
4:
2 subunit cDNA ratio injected into the oocytes (Fig.
1). The largest inward currents are
observed in oocytes injected with
4 and
2 subunit cDNAs in the
1:1 ratio. At both the 1:9 and 9:1
:
ratios, amplitudes of inward
currents evoked by 300 µM ACh are reduced significantly (t test; p < 0.001). Some oocytes (9 of 54)
that did not respond to ACh at all, most likely because the cDNA was
not properly injected into the nucleus, were excluded from the
analysis. The results show that, after injection of the
4 and
2
subunit cDNAs in extreme ratios, while maintaining the total cDNA
injected approximately constant, inward current amplitudes are reduced.
This indicates that after injection of cDNAs in the
:
ratios of
1:9 and 9:1, response amplitude is limited by the availability of
and
subunits, respectively. After injection of the same total
amounts of either cDNA encoding the
4 subunit only or cDNA encoding
the
2 subunit only, oocytes did not respond to 300 µM
ACh (results not shown). This demonstrates that
4 and
2 subunits
do not form functional homopentameric nAChRs. The absence of
ACh-induced inward current also shows that
4 and
2 do not form
functional heteromeric nAChRs with native nAChR subunits that possibly
are present in X. laevis oocytes (Buller and
White, 1990
).
|
Effects of
4:
2 ratio on the agonist concentration-effect
curve.
Agonist concentration-effect curves were obtained from
oocytes expressing
4
2 nAChRs after the injection of subunit cDNAs in the
:
ratios of 1:9, 1:1, and 9:1. Examples of ACh-induced ion
currents are shown in Fig. 2A for the
different subunit cDNA ratios tested.
|
:
ratios, inward current decay in the absence of overt ion
channel block (i.e., for responses evoked by superfusion with 300 µM ACh) was quantified by dividing the inward current at
the end of ACh application (t = 10 sec) by the peak inward current. The inward current decayed to 61 ± 16% (5 oocytes) of the peak value for the 1:1
:
ratio, to 51 ± 11% (7 oocytes) for the 9:1
:
ratio, and to 82 ± 4% (14 oocytes)
for the 1:9
:
ratio. The degree of inward current decay at the
1:9 ratio differs significantly from that at the 1:1 and 9:1
:
ratios (t test; p < 0.05).
For each of the
:
subunit cDNA ratios investigated, the
amplitudes of peak inward current evoked by superfusion of 0.1 µM to 10 mM ACh were normalized to those of 1 mM ACh-induced inward currents in three oocytes. Two-way
ANOVA of the results obtained with ACh at concentrations of
1
mM showed that the data for the 1:1 and 9:1
:
ratios
cannot be distinguished statistically (F1,28 = 0.1441, p = 0.71) and that both sets of data differ
statistically from the data obtained for the 1:9 ratio
(p < 0.001). Concentration-effect curves of
ACh were fitted for each
:
ratio. The data of the 1:1 and 9:1
:
ratios are closely approximated by one-component sigmoidal
curves (Fig. 2B). In all cases, data measured at the higher ACh
concentrations, which induce ion channel block, were excluded from the
curve-fitting procedure. For the 1:9
:
ratio, the
concentration-effect curve shows a striking shift toward lower agonist
concentrations, and the slope of the data is markedly reduced. Together
with the inflection point in the data observed between 10 and 100 µM ACh (Fig. 2B), this suggests the presence of two
components with distinct sensitivities to the agonist. A one-component
concentration-effect curve did not fit the data adequately as indicated
by analysis of the residuals (run test; p < 0.05). The
data obtained for concentrations of <1 mM ACh could be
fitted by a two-component concentration-effect curve only with fixed
values of the slope factors. EC50 values of 1.74 and 144 µM ACh and Emax
values of 85% and 43%, respectively, were obtained when the slope
factors were set to 1. Due to the small number of data available for
curve fitting at high agonist concentrations, reliable estimates for
the less-sensitive component cannot be obtained. A one-component
concentration-effect curve fitted to the data obtained with ACh at
concentrations of
30 µM yielded EC50, nH, and
Emax values of 1.84 µM ACh, 1.05, and 89.3%, respectively (Table
1). The two concentration-effect curves
both seem to fit the more-sensitive component accurately (Fig. 2B). The
large Emax value of the more-sensitive
component of the concentration-effect curve indicates that the majority
of nAChRs expressed at the 1:9
:
cDNA ratio are highly sensitive
to ACh. Oocytes injected with cDNAs encoding
3 and
2, or
4 and
4, in a 1:9
:
ratio did not show enhanced sensitivity to ACh
compared with oocytes injected with these subunit cDNAs in a 1:1
:
ratio (not shown).
|
4 and
2 subunit cDNAs
injected, two distinct types of functional nAChRs are expressed in
oocytes with ~30-fold difference in sensitivity to ACh.
Effects of
4:
2 ratio on the antagonist concentration-effect
curve.
Concentration-effect curves for the competitive nAChR
antagonist d-TC also were obtained from oocytes expressing
4
2
nAChRs after the injection of subunit cDNAs in the
:
ratios of
1:9, 1:1, and 9:1. Examples of ion currents evoked by superfusion of 300 µM ACh in the absence and the presence of various
concentrations of d-TC are shown in Fig.
3A for the different
subunit cDNA ratios tested. d-TC was superfused
4 min before an
ACh-induced current was recorded in the presence of d-TC. For three
oocytes of each
:
subunit cDNA ratio tested, peak inward current
amplitudes were normalized to those of control inward currents evoked
by 300 µM ACh in the same oocyte. The normalized data are
plotted against d-TC concentration in Fig. 3A. ANOVA of the inhibitory effects of d-TC showed that the data for the different subunit cDNA
ratios all are statistically different at the p < 0.001 level. For the 9:1 and 1:1
:
subunit cDNA ratios, data
could be fitted by a one-component concentration-effect curve. However,
for the 1:9 ratio, the concentration dependence of inhibition by d-TC seemed to be biphasic, and fit was significantly improved by fitting a
sum of two concentration-effect curves to the data (Fig. 3A). Estimated
values of the parameters of the fitted concentration-effect curves are
presented in Table 2. The inhibitory
effects of d-TC on
4
2 nAChRs at the 1:1
:
ratio also were
investigated at a holding potential of
40 mV (result not shown). The
IC50 value for d-TC of 0.91 ± 0.48 µM (three oocytes) at
40 mV could not be distinguished
from that obtained at the holding potential of
80 mV. Thus, it seems
that voltage-dependent ion channel block, observed previously for
muscle-type nAChRs at high concentrations of d-TC (Colquhoun et
al., 1979
), does not strongly contribute to the inhibitory effects
of d-TC observed at concentrations of
10 µM.
|
|
:
ratio.
Inward currents were evoked by superfusion with 10 µM ACh
to selectively activate the more sensitive nAChRs. The data on the
inhibition of 10 µM ACh-induced inward current by d-TC
are well fitted by a one-component concentration-effect curve (Fig. 3B)
with an IC50 value (see Table 2) that cannot be
distinguished from that of the more-sensitive component of inhibition
of 300 µM ACh-evoked responses (t test;
p = 0.11). Comparison of the inhibition curves of d-TC
obtained with 10 and 300 µM ACh (Fig. 3B) shows that at
the 1:9
:
ratio, two subtypes of nAChRs are expressed: one
subtype more sensitive to ACh and one less sensitive to d-TC than the
receptors expressed at the other
:
ratios, and a second subtype
with low sensitivity to ACh and very low sensitivity to d-TC. The
IC50 of d-TC shifts toward higher concentrations with decreasing
:
ratio (ANOVA; p values < 0.001), and the IC50 values of d-TC obtained at
the different
:
ratios differ statistically (t test;
p values < 0.01; Table 2).
To investigate the mechanism of inhibition of the component of
nAChR-mediated ion current with a very low sensitivity to d-TC observed
at the 1:9
:
ratio, oocytes were continuously superfused with 30 µM d-TC to eliminate the more-sensitive component (see Fig. 3A). Inward currents evoked by superfusion with 100 µM ACh in the presence of 30 µM d-TC, which
represent the less-sensitive component, and after preexposure to 330 µM d-TC for 4 min were recorded to establish low affinity
inhibition. Inward currents evoked at a holding potential of
60 mV
were reduced by 81.7 ± 1.6% (three oocytes) by increasing the
concentration of d-TC from 30 to 330 µM (Fig.
4A). In the same oocytes, low affinity
inhibition at a holding potential of
100 mV (Fig. 4B) amounted to
86.9 ± 2.4%. The small difference is statistically significant
(t test; p < 0.05), indicating some voltage
dependence of the inhibitory effect. Inward currents evoked by
coapplication of 100 µM ACh and 330 µM d-TC
show a reduction in the peak amplitude and a rapid further onset of
inhibition by d-TC during the response (Fig. 4C). At the steady level
of inhibition, which was achieved within a few seconds, the amount of
reduction of the inward current is the same as that obtained with
preexposure to d-TC. The apparent absence of kinetic effects after
preexposure to d-TC (Fig. 4, A and B) and the absence of a pronounced
tail current on removal of the high concentration of d-TC (Fig. 4C)
indicate that ion channel opening is not required for the inhibitory
effect. Together with the weak voltage dependence of inhibition, these
results indicate that even at high concentrations, d-TC does not cause significant ion channel block.
|
Effects of nAChR expression level on agonist and antagonist
sensitivities.
Because it cannot be excluded that nAChR expression
level by itself affects agonist and antagonist sensitivity, experiments were performed on oocytes injected with diluted 1:1
:
cDNA
solution at constant injection volume. In oocytes injected with a
10-fold diluted cDNA solution, superfusion with 1 mM ACh
did not evoke detectable (>5 nA) inward currents (10 oocytes). This
indicates that nAChR expression is greatly reduced, consistent with
previous results on the expression level of chick
4
2 nAChRs in
oocytes (Bertrand et al., 1991
). After the injection with a
4-fold diluted cDNA solution, ACh-induced ion currents were evoked,
which were smaller than those obtained before but had a similar shape
(Fig. 5A). The inward current decay at
the end of ACh application amounted to 48 ± 15% (five oocytes)
of the peak current. This cannot be distinguished from the degree of
decay at the higher expression level (t test;
p = 0.24) but differs from the degree of inward current
decay obtained for the 1:9
:
ratio (p < 0.01). Agonist and antagonist sensitivities were determined from six
oocytes with a maximal peak inward current amplitude of 398 ± 82 nA (i.e., in the same range as with the 1:9 and 9:1
:
ratios).
The results obtained with ACh at concentrations of
1 mM
from oocytes with high and low level nAChR expression at the 1:1
:
ratio are not identical (two-way ANOVA;
F1,28 = 7.556, p = 0.01).
Although the fitted concentration-effect curve of ACh (Fig. 5A, Table
1) shows a small shift toward higher concentrations, the
EC50 and nH
values cannot be distinguished from those obtained for the higher
expression level (t test; p > 0.08). The
concentration-effect curve of d-TC at the low expression level (Fig.
5B, Table 2) cannot be distinguished statistically from that obtained
with the high expression level at the 1:1
:
ratio (two-way ANOVA;
F1,20 = 2.290, p = 0.15). However, this curve differs from the inhibition curves obtained for the
9:1 and 1:9
:
ratios (two-way ANOVA; p < 0.001).
Further statistical analysis showed that the expression level does not influence the IC50 value of d-TC
(t-test; p = 0.33) and that regardless of
expression level, distinct IC50 values are
obtained for the different subunit ratios (t test;
p values < 0.01). From the combined results, it is
concluded that reducing the nAChR expression level causes only a small
decrease in ACh sensitivity, which is opposite the 30-fold increase in
sensitivity observed at the 1:9
:
ratio.
|
| |
Discussion |
|---|
|
|
|---|
Differences in agonist and antagonist sensitivities of receptors
expressed in oocytes after injection of cDNAs coding for the
4 and
2 subunits at 1:9, 1:1, and 9:1 ratios demonstrate heterogeneity of
4
2 nAChRs. The results show that combinations of
4 and
2
subunits form four pharmacologically distinct subtypes of heteromeric
nAChRs. Two subtypes of nAChRs, which are distinguished for the 1:1 and
9:1
:
ratios, have similar low sensitivities to ACh
(EC50
60 µM) but distinct
sensitivities to d-TC (IC50 = 0.20 and 0.53 µM, respectively). Two additional, distinct subtypes of
nAChRs are formed at the 1:9
:
ratio: one subtype with markedly enhanced sensitivity to ACh (EC50 = 1.8 µM) and decreased sensitivity to d-TC
(IC50 = 2.04 µM), and a second
subtype with low sensitivity to ACh and very low sensitivity to d-TC
(IC50 = 163 µM). Heterogeneity of
neuronal nAChRs has been suggested before from the observation that the
occurrence of distinct single-channel conductances in oocytes
expressing neuronal-type nAChRs depends on
:
ratio (Papke et al., 1989
).
The effects of varying
:
ratio on the properties of nAChRs were
investigated in oocytes injected with approximately equal total amounts
of cDNA (2.5-2.8 ng/oocyte). The reduced response amplitudes at the
1:9 and 9:1 ratios indicate that the availability of
and
subunits limits functional receptor expression at these cDNA ratios
(Fig. 1). A comparison of the properties of nAChRs expressed at a
reduced level for the 1:1
:
ratio (Fig. 5) with those of nAChRs
expressed at the 1:9 and 9:1 ratios (Figs. 2, 3) demonstrates that the
30-fold difference in sensitivity to ACh, the shift in sensitivity to
d-TC, and the differences in the degree of inward current decay are due
to changes in subunit ratio and not to differences in expression level.
The properties of ligand-gated ion channels depend on subunit
composition. Combining
4 and
2 subunits into a pentameric nAChR
protein theoretically can yield eight distinct subtypes of the
4
2
nAChR. The two homopentameric
4 and
2 nAChRs are not expressed or
are not functional. Six heteropentameric subunit assemblies remain:
four
:
subunit stoichiometries (1:4, 2:3, 3:2, and 4:1) and two
alternative subunit arrangements for the 2:3 and the 3:2
stoichiometries (Fig. 6). Of these six,
at least four have been demonstrated to form pharmacologically
distinct, functional nAChRs. This implies that functional
4
2
nAChRs are formed with other than the 2:3
:
subunit
stoichiometry.
|
Functional heteropentameric neuronal nAChRs presumably contain two
binding sites for ACh located at the 
interfaces (Luetje and
Patrick, 1991
; Bertrand and Changeux, 1995
). However, only two of all
possible subunit assemblies contain two 
interfaces (Fig. 6). The
conclusion that four distinct types of
4
2 nAChRs are formed
implies that agonist binding at one or more of the 
, 
, or

subunit interfaces may be involved in ion channel activation or
that binding of a single agonist molecule is sufficient to activate
4
2 nAChRs. The latter would be consistent with the results
because the slopes of the concentration-effect curves of ACh all are
close to 1 (Table 1). Shallow slopes of the concentration-effect curve
of ACh on
4
2 nAChRs generally are reported: in oocyte studies,
0.81 for rat
4
2 (Stafford et al., 1994
), 1.5 for chick
4
2 (Bertrand et al., 1990
), and 1.02 for human
4
2 (Chavez-Noriega et al., 1997
), and in human
embryonic kidney 293 cells, 1.2 for human
4
2 (Buisson and
Bertrand, 1998
). In
7 nAChRs, 
interfaces contribute to ligand
binding. This homopentameric nAChR seems to contain five binding sites
for the competitive antagonist methyllycaconitine (Palma et
al., 1996
). The
7 subunit contains the principal component (loops A-C) as well as the highly conserved residue Trp54, which constitutes part of a complementary component of the proposed agonist
binding site. These two components of the agonist binding site in
7
are thought to be equivalent to those contained by the muscle-type
1
and the
and
nAChR subunits, respectively. The
4 subunit
contains the principal component of the agonist binding site as well as
a tryptophan residue homologous to Trp54 in
7. The
2 subunit
contains the complementary component of the agonist binding site as
well as loops A and B of the principal component (Corringer et
al., 1995
). The homology among
4,
2, and
7 suggests that

, 
, or 
subunit interfaces also might form functional
agonist or antagonist binding sites. The shift in agonist sensitivity
seems to be specific for the
4
2 nAChRs because it is not observed
when
3
2 and
4
4 nAChRs are expressed at 1:1 and 1:9
:
cDNA ratios (Luetje and Patrick, 1991
). This indicates that neither

nor 
subunit interfaces are directly involved in the
change in sensitivity to ACh.
The results show that the inhibitory effects of d-TC on
4
2 nAChRs
are not due to ion channel block. Low concentrations of d-TC have been
shown to inhibit neuronal nAChRs by a competitive interaction before
(Bertrand et al., 1990
), but the exact nature of the
inhibitory effect of the very high concentrations of d-TC on nAChRs at
the 1:9
:
ratio remains to be investigated. However, the four
distinct IC50 values for d-TC do not necessarily
imply that
4
2 nAChRs contain four distinct binding sites for
d-TC. Apart from the component with very low sensitivity, the effects of d-TC consist of monophasic inhibition curves with shallow slopes, which gradually shift to higher concentrations with decreasing
:
ratio (Fig. 3; Table 2). This indicates that high and low affinity
sites differentially contribute to the inhibitory effect of d-TC and
cause shift of the IC50. Three two-component
inhibition curves of d-TC were simultaneously fitted to the three sets
of data obtained for the 9:1, 1:1, and 1:9 (10 µM ACh)
:
ratios. Hill coefficients were set to 1, and the
IC50 values of d-TC and the relative proportions
of the two effects of d-TC were estimated. This yielded
IC50 values of 0.16 and 4.3 µM
d-TC, regardless of
:
ratio. The estimated proportions of the
more- and less-sensitive components changed with
:
ratio. The
size of the more sensitive component decreased from 88% at the 9:1
:
ratio to 62% and further to 26% at the 1:1 and 1:9
:
ratios, respectively. The curves fitted according to the two-component
inhibition model (Fig. 3) demonstrate that for
4
2 nAChRs, at
least two sites with an estimated 27-fold difference in sensitivity to
d-TC are required to account for the inhibitory effects observed. This
is in the same order of magnitude as the 15- and 27-fold differences in
sensitivity of
1
1
and
1
1
muscle-type nAChRs expressed
in fibroblasts and human embryonic kidney 293 cells to d-TC and
dimethyl-d-TC, respectively (Blount and Merlie, 1989
; Sine, 1993
). The
shift in the inhibition curve of d-TC can be accounted for by assuming a minimum of two distinct sites for d-TC. The differential contribution of these sites to the inhibitory effect at different
:
ratios suggests that they are located on distinct subtypes of
4
2 nAChRs. The possibility that the high and low affinity components reflect effects of d-TC on a single population of receptors is highly unlikely.
If the two sites would be present on the same nAChR, only the
inhibition caused by the binding of d-TC to the high affinity site
would have been detected. Some receptors seem to lack both sites
because they are affected by d-TC at very high concentrations only.
The results with d-TC indicate that two subtypes of
4
2 nAChRs may
be distinguished in oocytes injected with cDNAs at the 1:1
:
ratio. In oocytes injected with cRNAs or cDNAs encoding chick
4 and
2 subunits at a 1:1 ratio, nAChRs seem to have a 2:3
:
stoichiometry (Anand et al., 1991
; Cooper et al.,
1991
). The current and the previous results are compatible when two
subunit arrangements, which are possible for the 2:3
:
stoichiometry (Fig. 6), are formed and have distinct sensitivities to
d-TC. The diverging results on the number of conductance levels of
single ion channels formed by
4
2 nAChRs at the 1:1
:
ratio
provide no conclusive evidence on
4
2 nAChR heterogeneity (Papke
et al., 1989
; Cooper et al., 1991
; Charnet
et al., 1992
; Kuryatov et al., 1997
).
Single-channel properties of
4
2 nAChRs at other than the 1:1
:
ratio have not been reported. Whether the arrangement of
subunits within a receptor affects single-channel properties remains
unknown. The current results demonstrate that changing the relative
abundance of subunits expressed by changing the cDNA ratio leads to
alternative subunit assemblies with sensitivities to ACh and d-TC
distinct from those obtained for nAChRs at the 1:1
:
ratio.
The ratios of subunit cDNAs used in the current study for the
expression of distinct subtypes of
4
2 nAChRs in oocytes are in
the same order of magnitude as those found in brain. The ratio of
4:
2 mRNA levels in various regions of rat brain ranges between 1:1 and 1:15 (Liu et al., 1996
). For
-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid-type glutamate
receptors, it has been shown that the number of glutamic acid type 2 receptor subunits incorporated depends on the relative abundance of the
glutamic acid type 2 receptor subunit expressed in oocytes as well as
in rat hippocampal interneurons. The resulting differences in subunit
stoichiometry of native and heterologously expressed
-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors are
associated with changes in channel function (Washburn et
al., 1997
). Although it was assumed previously that nAChRs have a
fixed subunit stoichiometry, the current results show that
4
2
nAChR subunit stoichiometry also can be changed by altering the
relative abundance of
and
subunits. Thus, changing the subunit
stoichiometry of receptors by selective up- or down-regulation of the
expression level of specific subunits seems to be a more general
mechanism to tune the properties of ligand-gated ion channels. The
current observation that agonist and antagonist sensitivities depend on
subunit stoichiometry provides a rational basis for the existence of
heteromeric neuronal nAChRs.
| |
Acknowledgments |
|---|
We thank Dr. Jim Patrick (Baylor College of Medicine, Houston, TX) for donating the cDNA clones of nAChR subunits, Kees Koster (Hubrecht Laboratory, Utrecht, The Netherlands) for supplying X. laevis oocytes, John Rowaan for taking care of the frogs in our laboratory, and Ing. Aart de Groot for excellent technical assistance.
| |
Footnotes |
|---|
Received May 18, 1998; Accepted September 14, 1998
This work was financially supported by Netherlands Organization for Scientific Research (NWO) Grant 903-42-011.
Send reprint requests to: Dr. R. Zwart, Research Institute of Toxicology, Utrecht University, P.O. Box 80.176, NL-3508 TD Utrecht, The Netherlands. E-mail: r.zwart{at}ritox.vet.uu.nl
| |
Abbreviations |
|---|
nAChR, nicotinic acetylcholine receptor; ACh, acetylcholine; ANOVA, analysis of variance; d-TC, d-tubocurarine; HEPES, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid.
| |
References |
|---|
|
|
|---|
4
2 neuronal nicotinic acetylcholine receptor.
Mol Pharmacol
53:
555-563
,
, and
subunit RNAs are a consequence of endogenous oocyte gene expression.
Mol Pharmacol
37:
423-428[Abstract].
4
2 neuronal nicotinic acetylcholine receptors expressed in Xenopus oocytes.
J Physiol (Lond)
450:
375-394
2
2, h
2
4, h
3
2, h
3
4, h
4
2, h
4
4 and h
7 expressed in Xenopus oocytes.
J Pharmacol Exp Ther
280:
346-356
7 homooligomeric receptor.
J Biol Chem
270:
11749-11752
4 and
2 subunits and is up-regulated by chronic nicotine treatment.
Mol Pharmacol
41:
31-37[Abstract].
4
2 nicotinic acetylcholine receptors.
J Neurosci
17:
9035-9047
4 and
2 subunit genes in various regions of rat brain.
NeuroReport
7:
1645-1649[Medline].
- and
-subunits contribute to the agonist sensitivity of neuronal nicotinic acetylcholine receptors.
J Neurosci
11:
837-845[Abstract].
7 receptor expressed in Xenopus oocytes presents five putative binding sites for methyllycaconitine.
J Physiol (Lond)
491:
151-161
4 subunit in determining the kinetic properties of rat neuronal nicotinic acetylcholine
3 receptors.
J Physiol (Lond)
440:
95-112
subunit of neuronal nicotinic acetylcholine receptors is a determinant of the affinity for substance P inhibition.
Mol Pharmacol
45:
758-762[Abstract].
2,
3,
4, and
2 neuronal nicotinic receptor subunit mRNAs in the central nervous system: a hybridization histochemical study in the rat.
J Comp Neurol
284:
314-335[Medline].
3
2 and
3
4 neuronal nicotinic receptors expressed in Xenopus oocytes by flufenamic acid and niflumic acid.
J Neurosci
15:
2168-2178[Abstract].This article has been cited by other articles:
![]() |
T. D. McClure-Begley, N. M. King, A. C. Collins, J. A. Stitzel, J. M. Wehner, and C. M. Butt Acetylcholine-Stimulated [3H]GABA Release from Mouse Brain Synaptosomes is Modulated by {alpha}4{beta}2 and {alpha}4{alpha}5{beta}2 Nicotinic Receptor Subtypes Mol. Pharmacol., April 1, 2009; 75(4): 918 - 926. [Abstract] [Full Text] [PDF] |
||||
![]() |
N. B. Fedorov, L. C. Benson, J. Graef, P. M. Lippiello, and M. Bencherif Differential Pharmacologies of Mecamylamine Enantiomers: Positive Allosteric Modulation and Noncompetitive Inhibition J. Pharmacol. Exp. Ther., February 1, 2009; 328(2): 525 - 532. [Abstract] [Full Text] [PDF] |
||||
![]() |
B. Lamotte d'Incamps and P. Ascher Four Excitatory Postsynaptic Ionotropic Receptors Coactivated at the Motoneuron-Renshaw Cell Synapse J. Neurosci., December 24, 2008; 28(52): 14121 - 14131. [Abstract] [Full Text] [PDF] |
||||
![]() |
M. Moroni, R. Vijayan, A. Carbone, R. Zwart, P. C. Biggin, and I. Bermudez Non-Agonist-Binding Subunit Interfaces Confer Distinct Functional Signatures to the Alternate Stoichiometries of the {alpha}4{beta}2 Nicotinic Receptor: An {alpha}4-{alpha}4 Interface Is Required for Zn2+ Potentiation J. Neurosci., July 2, 2008; 28(27): 6884 - 6894. [Abstract] [Full Text] [PDF] |
||||
![]() |
C. Gotti, M. Moretti, N. M. Meinerz, F. Clementi, A. Gaimarri, A. C. Collins, and M. J. Marks Partial Deletion of the Nicotinic Cholinergic Receptor {alpha}4 or {beta}2 Subunit Genes Changes the Acetylcholine Sensitivity of Receptor-Mediated 86Rb+ Efflux in Cortex and Thalamus and Alters Relative Expression of {alpha}4 and {beta}2 Subunits Mol. Pharmacol., June 1, 2008; 73(6): 1796 - 1807. [Abstract] [Full Text] [PDF] |
||||
![]() |
R. Zwart, A. L. Carbone, M. Moroni, I. Bermudez, A. J. Mogg, E. A. Folly, L. M. Broad, A. C. Williams, D. Zhang, C. Ding, et al. Sazetidine-A Is a Potent and Selective Agonist at Native and Recombinant {alpha}4{beta}2 Nicotinic Acetylcholine Receptors Mol. Pharmacol., June 1, 2008; 73(6): 1838 - 1843. [Abstract] [Full Text] [PDF] |
||||
![]() |
N. P. Barrera, J. Betts, H. You, R. M. Henderson, I. L. Martin, S. M. J. Dunn, and J. M. Edwardson Atomic Force Microscopy Reveals the Stoichiometry and Subunit Arrangement of the {alpha}4{beta}3{delta} GABAA Receptor Mol. Pharmacol., March 1, 2008; 73(3): 960 - 967. [Abstract] [Full Text] [PDF] |
||||
![]() |
S. H. Hadley and J. Amin Rat {alpha}6beta2{delta} GABAA receptors exhibit two distinct and separable agonist affinities J. Physiol., June 15, 2007; 581(3): 1001 - 1018. [Abstract] [Full Text] [PDF] |
||||
![]() |
L. Tapia, A. Kuryatov, and J. Lindstrom Ca2+ Permeability of the ({alpha}4)3(beta2)2 Stoichiometry Greatly Exceeds That of ({alpha}4)2(beta2)3 Human Acetylcholine Receptors Mol. Pharmacol., March 1, 2007; 71(3): 769 - 776. [Abstract] [Full Text] [PDF] |
||||
![]() |
M. J. Marks, P. Whiteaker, and A. C. Collins Deletion of the {alpha}7, beta2, or beta4 Nicotinic Receptor Subunit Genes Identifies Highly Expressed Subtypes with Relatively Low Affinity for [3H]Epibatidine Mol. Pharmacol., September 1, 2006; 70(3): 947 - 959. [Abstract] [Full Text] [PDF] |
||||
![]() |
M. Moroni, R. Zwart, E. Sher, B. K. Cassels, and I. Bermudez {alpha}4beta2 Nicotinic Receptors with High and Low Acetylcholine Sensitivity: Pharmacology, Stoichiometry, and Sensitivity to Long-Term Exposure to Nicotine Mol. Pharmacol., August 1, 2006; 70(2): 755 - 768. [Abstract] [Full Text] [PDF] |
||||
![]() |
C. A. Briggs, E. J. Gubbins, M. J. Marks, C. B. Putman, R. Thimmapaya, M. D. Meyer, and C. S. Surowy Untranslated Region-Dependent Exclusive Expression of High-Sensitivity Subforms of {alpha}4beta2 and {alpha}3beta2 Nicotinic Acetylcholine Receptors Mol. Pharmacol., July 1, 2006; 70(1): 227 - 240. [Abstract] [Full Text] [PDF] |
||||
![]() |
P. J. Groot-Kormelink, S. Broadbent, M. Beato, and L. G. Sivilotti Constraining the Expression of Nicotinic Acetylcholine Receptors by Using Pentameric Constructs Mol. Pharmacol., February 1, 2006; 69(2): 558 - 563. [Abstract] [Full Text] [PDF] |
||||
![]() |
M. M. White Pretty Subunits All in a Row: Using Concatenated Subunit Constructs to Force the Expression of Receptors with Defined Subunit Stoichiometry and Spatial Arrangement Mol. Pharmacol., February 1, 2006; 69(2): 407 - 410. [Abstract] [Full Text] [PDF] |
||||
![]() |
A. M. Person, K. L. Bills, H. Liu, S. K. Botting, J. Lindstrom, and G. B. Wells Extracellular Domain Nicotinic Acetylcholine Receptors Formed by {alpha}4 and {beta}2 Subunits J. Biol. Chem., December 2, 2005; 280(48): 39990 - 40002. [Abstract] [Full Text] [PDF] |
||||
![]() |
P. V. Plazas, E. Katz, M. E. Gomez-Casati, C. Bouzat, and A. B. Elgoyhen Stoichiometry of the {alpha}9{alpha}10 Nicotinic Cholinergic Receptor J. Neurosci., November 23, 2005; 25(47): 10905 - 10912. [Abstract] [Full Text] [PDF] |
||||
![]() |
N. O. Rodrigues-Pinguet, T. J. Pinguet, A. Figl, H. A. Lester, and B. N. Cohen Mutations Linked to Autosomal Dominant Nocturnal Frontal Lobe Epilepsy Affect Allosteric Ca2+ Activation of the {alpha}4{beta}2 Nicotinic Acetylcholine Receptor Mol. Pharmacol., August 1, 2005; 68(2): 487 - 501. [Abstract] [Full Text] [PDF] |
||||
![]() |
H. C. Ben-Ami, L. Yassin, H. Farah, A. Michaeli, M. Eshel, and M. Treinin RIC-3 Affects Properties and Quantity of Nicotinic Acetylcholine Receptors via a Mechanism That Does Not Require the Coiled-coil Domains J. Biol. Chem., July 29, 2005; 280(30): 28053 - 28060. [Abstract] [Full Text] [PDF] |
||||
![]() |
B. S. Khakh, J. A. Fisher, R. Nashmi, D. N. Bowser, and H. A. Lester An Angstrom Scale Interaction between Plasma Membrane ATP-Gated P2X2 and {alpha}4{beta}2 Nicotinic Channels Measured with Fluorescence Resonance Energy Transfer and Total Internal Reflection Fluorescence Microscopy J. Neurosci., July 20, 2005; 25(29): 6911 - 6920. [Abstract] [Full Text] [PDF] |
||||
![]() |
X.-Q. Ren, S.-B. Cheng, M. W. Treuil, J. Mukherjee, J. Rao, K. H. Braunewell, J. M. Lindstrom, and R. Anand Structural Determinants of {alpha}4{beta}2 Nicotinic Acetylcholine Receptor Trafficking J. Neurosci., July 13, 2005; 25(28): 6676 - 6686. [Abstract] [Full Text] [PDF] |
||||
![]() |
H. Tsuneki, Y. You, N. Toyooka, S. Kagawa, S. Kobayashi, T. Sasaoka, H. Nemoto, I. Kimura, and J. A. Dani Alkaloids Indolizidine 235B', Quinolizidine 1-epi-207I, and the Tricyclic 205B are Potent and Selective Noncompetitive Inhibitors of Nicotinic Acetylcholine Receptors Mol. Pharmacol., October 1, 2004; 66(4): 1061 - 1069. [Abstract] [Full Text] [PDF] |
||||
![]() |
G. Y. Lopez-Hernandez, J. Sanchez-Padilla, A. Ortiz-Acevedo, J. Lizardi-Ortiz, J. Salas-Vincenty, L. V. Rojas, and J. A. Lasalde-Dominicci Nicotine-induced Up-regulation and Desensitization of {alpha}4{beta}2 Neuronal Nicotinic Receptors Depend on Subunit Ratio J. Biol. Chem., September 3, 2004; 279(36): 38007 - 38015. [Abstract] [Full Text] [PDF] |
||||
![]() |
R. Nashmi, M. E. Dickinson, S. McKinney, M. Jareb, C. Labarca, S. E. Fraser, and H. A. Lester Assembly of {alpha}4{beta}2 Nicotinic Acetylcholine Receptors Assessed with Functional Fluorescently Labeled Subunits: Effects of Localization, Trafficking, and Nicotine-Induced Upregulation in Clonal Mammalian Cells and in Cultured Midbrain Neurons J. Neurosci., December 17, 2003; 23(37): 11554 - 11567. [Abstract] [Full Text] [PDF] |
||||
![]() |
K G Paradiso and J. H. Steinbach Nicotine is highly effective at producing desensitization of rat {alpha}4{beta}2 neuronal nicotinic receptors J. Physiol., December 15, 2003; 553(3): 857 - 871. [Abstract] [Full Text] [PDF] |
||||
![]() |
Y. Zhou, M. E. Nelson, A. Kuryatov, C. Choi, J. Cooper, and J. Lindstrom Human {alpha}4{beta}2 Acetylcholine Receptors Formed from Linked Subunits J. Neurosci., October 8, 2003; 23(27): 9004 - 9015. [Abstract] [Full Text] [PDF] |
||||
![]() |
M. E. Nelson, A. Kuryatov, C. H. Choi, Y. Zhou, and J. Lindstrom Alternate Stoichiometries of alpha 4beta 2 Nicotinic Acetylcholine Receptors Mol. Pharmacol., February 1, 2003; 63(2): 332 - 341. [Abstract] [Full Text] [PDF] |
||||
![]() |
S. S Khiroug, P. C Harkness, P. W Lamb, S. N Sudweeks, L. Khiroug, N. S Millar, and J. L Yakel Rat nicotinic ACh receptor {alpha}7 and {beta}2 subunits co-assemble to form functional heteromeric nicotinic receptor channels J. Physiol., April 15, 2002; 540(2): 425 - 434. [Abstract] [Full Text] [PDF] |
||||
![]() |
B. Buisson and D. Bertrand Chronic Exposure to Nicotine Upregulates the Human {alpha}4{beta}2 Nicotinic Acetylcholine Receptor Function J. Neurosci., March 15, 2001; 21(6): 1819 - 1829. [Abstract] [Full Text] [PDF] |
||||
![]() |
A. Figl and B. N Cohen The {beta} subunit dominates the relaxation kinetics of heteromeric neuronal nicotinic receptors J. Physiol., May 1, 2000; 524(3): 685 - 699. [Abstract] [Full Text] [PDF] |
||||
![]() |
S. S. Khiroug, P. C. Harkness, P. W. Lamb, S. N. Sudweeks, L. Khiroug, N. S. Millar, and J. L. Yakel Rat nicotinic ACh receptor {alpha}7 and {beta}2 subunits co-assemble to form functional heteromeric nicotinic receptor channels J. Physiol., February 22, 2002; (2002) 200101384. [Abstract] [PDF] |
||||
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||