From the Department of Pharmacology, University
College London, Gower St., London WC1E 6BT, United Kingdom and the
§ Department of Experimental Molecular Biology, Janssen
Research Foundation, Turnhoutseweg 30, B-2340 Beerse, Belgium
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ABSTRACT |
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We have investigated whether the neuronal
nicotinic subunit 3 can participate in the assembly of functional
recombinant receptors. Although
3 is expressed in several areas of
the central nervous system, it does not form functional receptors when
expressed heterologously together with an
or another
nicotinic
subunit. We inserted into the human
3 subunit a reporter mutation
(V273T), which, if incorporated into a functional receptor, would be
expected to increase its agonist sensitivity and maximum response to
partial agonists. Expressing the mutant
3V273T in
Xenopus oocytes together with both the
3 and the
4
subunits resulted in the predicted changes in the properties of the
resulting nicotinic receptor when compared with those of
3
4
receptors. This indicated that some of the receptors incorporated the
mutant
3 subunit, as part of a "triplet"
3
4
3 receptor.
The proportion of triplet receptors was dependent on the ratios of the
3:
4:
3 cRNA injected. We conclude that, like the related
5
subunit, the
3 subunit can form functional receptors only if
expressed together with both
and
subunits.
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INTRODUCTION |
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Molecular cloning has brought to light an unsuspected multitude of subunits that are the building blocks of ligand-gated ion channels. Our understanding of the functional importance of this diversity is still very incomplete. We need to know which subunits can co-assemble, which ones actually do so in native tissue, and whether the properties of the receptors assembled from different subunit combinations are different in a physiologically meaningful way (1).
A good example is that of the neuronal nicotinic acetylcholine
(ACh)1 receptors, for which
as many as 11 different subunits (named 2-
9 and
2-
4) have
been cloned. Heterologous expression has shown that, apart from
7,
8, and
9, which can form homomeric receptors, functional
receptors require the co-expression of an "
/
" pair of
subunits, i.e.
2-4 with
2 or
4 (2). The
6 subunit contributes to functional receptors if expressed with
2,
with
4 or, as a "triplet," with
3 and
4 (3, 4). Neither
5 nor
3 can form
/
"pair" receptors:
5 can form
functional triplet receptors with
3
2,
3
4, or
4
2
(detectable because of changes in agonist sensitivity, macroscopic
desensitization, and channel conductance (5-7)). Although the
3
subunit was discovered more than 9 years ago (8), its role remains
obscure. It could be that
3 is a transcribed pseudogene
(i.e. a non-functional gene) or that the
3 subunit
co-assembles into a functional nicotinic receptor only with another,
yet to be identified, subunit. Alternatively, it is conceivable that
3, like the
5 subunit, could form functional receptors only if
expressed in a triplet combination. This is supported by the high
similarity between the
5 and
3 subunit (80% amino acid sequence
similarity, i.e. identical amino acids and conservative
substitutions) and they have been classified in a separate group within
the neuronal nicotinic receptor family (9).
However, the number of potential triplet combinations that would have
to be screened is large, because 3 can be immunoprecipitated from at
least five different brain regions, where it is present in neurones
that express a variety of other subunits (2, 10, 11). Unless the
effects of
3 on receptor properties are as striking as those of
5, the range of tests to be carried out is likely to be extensive.
In addition, there may be differences in triplet receptor assembly
between oocytes and cell lines (12), e.g.
3
4 receptors
expressed in oocytes differ in their channel properties from
3
4
receptors expressed in HEK293 cells (13). Until it has been established
which of the heterologous expression systems is closer to native
neurones for each combination, it is desirable that these tests should
be carried out both in oocytes and in cell lines.
We have investigated which 3-containing triplet combinations
assemble by inserting into the
3 subunit a reporter mutation, V273T.
This mutation converts the hydrophobic residue in the middle of the
pore-lining second transmembrane domain (TM2) into a hydrophilic residue. In the nicotinic receptors in which this type of mutation has
been tested (
7 (14) and mouse muscle (15, 16)), in the
1
2
2
-aminobutyric acid receptor (17), and in the 5HT3 receptor (18), it resulted in a pronounced leftward shift of the
agonist concentration-response curve. This shift was found to increase
regularly with the number of mutated subunits incorporated (15) and is
likely to result from changes in the gating equilibrium constant due to
destabilization of the closed state, although there may also be a
contribution by the desensitized state becoming conducting (14).
We found that expressing 3V273T together with the
3
and
4 subunits in Xenopus oocytes changes the
pharmacological properties of the
3
4 recombinant nicotinic
receptor, suggesting that the
3V273T subunit is
incorporated into a functional
3
4
3V273T
receptor.
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EXPERIMENTAL PROCEDURES |
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Construction of cRNA for Oocyte Expression--
cDNAs for
the human 3,
3, and
4 (GenBankTM accession numbers
Y08418, Y08417, and Y08416, respectively), containing only coding
sequences and an added Kozak consensus sequence (GCCACC) immediately
upstream of the start codon (19), were subcloned into the pSP64GL
vector, which contains 5'- and 3'-untranslated Xenopus
-globin regions (20). The V273T mutation was inserted in
3 using
the QuickChangeTM Site-directed Mutagenesis Kit
(Stratagene; mutagenesis primer used:
5'-GTCGACAGAAAAGACTTCTTCGATAACGGAGAATGG-3'), and the full-length sequence was verified. All four cDNA/pSP64GL plasmids were
linearized immediately downstream of the 3'-untranslated
-globin
sequence, and cRNA was transcribed using the SP6 Mmessage Mmachine Kit
(Ambion). The quality and quantity were checked by RNA gel
electrophoresis and comparison with RNA concentration and size
markers.
Expression in Xenopus Oocytes--
Mature female Xenopus
laevis frogs were anesthetized by immersion in a 0.2% solution
(pH 5.6) of ethyl m-aminobenzoate (methanesulfonate, Tricaine, Sigma) and killed by decapitation and destruction of the
brain and the spinal cord. The ovarian lobes were dissected into small
clumps of 5-10 oocytes, treated with collagenase (Sigma IA; 245 collagen digestion units/ml) for 75 min, and defolliculated manually.
Healthy stage V-VI oocytes were selected for injection of cRNA coding
for the nicotinic subunits to be expressed (23 nl, 1.2 pg of cRNA in
total per oocyte). cRNA ratios were 1:1 for 3
4, 1:1:1 and 1:1:20
for
3
4 +
3V273T, and 1:1:20 for
3
4 +
3WT injections. The injection was carried out with a
Drummond Nanoject injector. Oocytes were incubated in Barth's solution
at 18 °C for 2 days, then stored at 4-6 °C until needed for
electrophysiological recording (up to 2 weeks later).
Electrophysiological Recording--
Current responses were
obtained by two-electrode voltage clamp recording at a holding
potential of 70 mV (Axoclamp 2B, Axon Instrument), with electrodes
filled with 3 M KCl. Agonists solutions (ACh chloride and
(
)-nicotine hydrogen tartrate, both from Sigma) were freshly prepared
in modified Ringer's solution from frozen aliquots of stock and
bath-applied (approximately 5 ml/min) to elicit inward currents which
were recorded on a chart for subsequent analysis. Agonist responses
were obtained at 5-min intervals, and a standard ACh concentration (50 µM) was applied every third response. The responses to 50 µM ACh were used to correct for rundown of response
amplitude during the experiment, by linear interpolation.
Solutions--
Barth's solution for oocyte culture had a
composition of (mM) NaCl (88), KCl (1), MgCl2
(0.82), CaCl2 (0.77), NaHCO3 (2.4), Tris-HCl
(15), with 50 units ml1 penicillin and 50 µg
ml
1 streptomycin, pH 7.4. The solution was sterilized by
filtration (0.22 µm pore filter, Millex-GV, Millipore).
Data Analysis-- A full concentration-response curve to ACh was obtained for each oocyte included in this study. Analysis of the results was carried out by fitting each concentration-response curve separately (by equally weighted least squares, CvFit program by David Colquhoun) with the Hill equation I = Imax(xnH/(EC50nH + xnH)) where Imax is the maximum response to the agonist, x the agonist concentration, nH the Hill coefficient, and EC50 the agonist concentration that produces 50% of the maximum response.
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RESULTS AND DISCUSSION |
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We have expressed in Xenopus oocytes the 3
4
combination alone or together with wild type
3 (
3WT)
or mutant
3 (
3V273T). Addition of cRNA for the
3WT subunit to
3 and
4 did not produce a
detectable shift in the ACh concentration-response curve (see Fig.
1 and Table
I), but when
3V273T was
added instead of the
3WT, a pronounced leftward shift in
the ACh concentration-response curve was observed. This suggests that
the mutated
3 subunit is incorporated into the expressed
receptor.
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The next question is, what proportion of the receptors expressed after
injection of 3,
4, and
3 do actually contain
3? The results
in Table I and Fig. 1 were all obtained by injection of cRNAs for these
subunits in a ratio of 1:1:20. We can obtain an estimate of the
proportion of current due to triplet receptors by fitting the ACh
concentration-response curves with the sum of two Hill equations, the
first of which is supposed to represent pair receptors and therefore
has its EC50 and Hill slope (nH) fixed at the values already determined in oocytes expressing
3
4 alone (180 µM and 1.81, respectively (Table I)). Fig.
1B shows four concentration-response curves from oocytes
injected with
3,
4, and
3V273T cRNAs in the ratio
1:1:1. The fit shown (see legend) gives an estimate of the fraction of
current carried by triplet channels as 61 ± 4.3%
(n = 4) with 2-unit likelihood intervals (roughly 95%
confidence) of 54% to 73%. The EC50 and
nH values estimated for the mutant triplet
receptor (2nd component in Fig. 1B) were 37.7 ± 3.6 µM and 1.31 ± 0.09 for the 1:1:1 injections with
3V273T, very close to the values obtained by a
two-component fit of the 1:1:20 injections (41.8 ± 6.5 µM, 1.22 ± 0.06). When the cRNAs were injected in
the ratio 1:1:20, fitting two components produced only a slight
improvement (0.5 log likelihood units) over a single component, so it
was not possible to estimate the fraction attributable to the second
component (for either
3WT or
3V273T), but
the results were consistent with a large proportion of the receptors
being triplets.
A change in gating for receptors containing the mutant subunit
would also be expected to change the agonist efficacy, the maximum open
probability, and therefore the maximum agonist response. Whether the
change in maximum response is big enough to be detected will depend on
whether efficacy increases or decreases and on the agonist efficacy in
the wild-type receptor. If the mutation shifts the equilibrium toward
the open state of the bound receptor (as in our case), efficacy should
increase, but an increase in the maximum agonist response would be
noticeable only if the wild-type maximum open probability was well
below 1, i.e. if the agonist is a partial agonist (21).
While little is known of the actual efficacy of nicotinic agonists on
neuronal receptors, nicotine is a good example of a possible partial
agonist on 3
4, as it elicits a maximum response which is only
44.7 ± 2.3% of that to ACh (see Fig.
2). The maximum response to nicotine (as
a fraction of the ACh maximum) is nearly doubled by the mutation,
fulfilling the prediction (see
3
4
3WT
versus
3
4
3V273T in Table I and in Fig. 2).
Clearly, changes in agonist channel block and in desensitization
(following the V273T mutation) may contribute to this effect, and
further work is necessary to exclude this possibility.
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We have shown that the 3 subunit can be incorporated into a
functional
3
4
3 recombinant nicotinic receptor. While
3 is expressed in several areas of the nervous system which lack
3 and
4, the
3 subunit is also abundant in sensory ganglia, which are
rich in
3 and
4 subunits. The nicotinic receptors of sensory ganglia (which are potential targets for nicotinic analgesia (22)) may
therefore have an
3
4
3 composition. The reporter mutation approach used here will allow rapid screening of other subunit combinations containing
3 and will establish which of these can form
functional receptors. In addition, the optimal cRNA ratios needed to
ensure that the majority of assembled receptors are triplets can be
determined. Once combination and optimal conditions have been
identified by expression of the mutant, the wild-type triplet can be
expressed heterologously to characterize the pharmacological and
biophysical properties of the resulting receptor. Furthermore, if in
neuronal nicotinic receptors, as in muscle receptors, the EC50 shift produced by the hydrophilic mutation in the
middle of TM2 is proportional to the number of mutated subunits (15, 16), this approach can also be extended to the question of the stoichiometry of triplet receptors. Finally, as the mid-TM2 Leu (or
Val) motif is present in all subunits belonging to the 4-transmembrane domain receptor superfamily, the method can readily be extended to
other receptor types.
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ACKNOWLEDGEMENTS |
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We thank John Wood, Armen Akopian, David Attwell, and Danny Huylebroeck for advice.
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FOOTNOTES |
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* This work was supported in part by a Medical Research Council grant (to D. C.).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.
¶ To whom correspondence should be addressed: Dept. of Pharmacology, The School of Pharmacy, 29/39 Brunswick Square, London WC1N 1AX, UK. Tel.: 44-171-753-5887; Fax: 44-171-753 5902; E-mail: sivilo{at}cua.ulsop.ac.uk.
1 The abbreviations used are: ACh, acetylcholine; EC50, the agonist concentration that produces 50% of the maximum response; Imax, the maximum response to the agonist; nH, the Hill coefficient; TM2, second transmembrane domain.
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REFERENCES |
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