Address correspondence to Lucia G. Sivilotti, Department of Pharmacology, University College London, Gower Street, London WC1E 6BT, United Kingdom. Fax: (44) 20-7679-7298; email: l.sivilotti{at}ucl.ac.uk
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ABSTRACT |
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Key Words: oocytes two-electrode voltage-clamp reporter mutation approach acetylcholine ion channels
Abbreviation used in this paper: nAChR, nicotinic acetylcholine receptor.
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INTRODUCTION |
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We set out to use tandem constructs to express neuronal nicotinic acetylcholine receptors (nAChRs), with a view ultimately to obtain efficient expression of complex heteromeric combinations and examined in detail if the assumptions of the technique were valid for the tandem constructs that gave the best expression.
Tandem constructs should be particularly useful for neuronal nAChR, which, in their native form, can contain as many as four different subunits within the same pentamer (Conroy and Berg, 1995; Forsayeth and Kobrin, 1997
). Functional recombinant neuronal nAChRs can however be obtained from simpler combinations, homomeric (one
subunit) or heteromeric (combinations of
and ß subunits; for review see Colquhoun et al., 2003
). Heterologous expression should in principle give good results for homomers, for heteromeric "pair" receptors (formed by a single
-type together with a single ß-type subunit), and for heteromeric "triplet" receptors provided the third subunit,
5 or ß3, cannot form pair receptors (Ramirez-Latorre et al., 1996
; Wang et al., 1996
; Groot-Kormelink et al., 1998
). However, it is difficult to see how the complex native neuronal nAChRs combinations can be reliably produced in vitro. Even for apparently simple "pair" combinations, such as
4ß2, nAChR stoichiometry depends on the expression system (Nelson et al., 2003
).
A technique for obtaining receptors with a defined composition would therefore be a major advance. In the simplest strategy used in the nicotinic superfamily, tandem constructs (i.e., two subunits connected by a linker) are expressed with the appropriate monomer to produce a "pure" receptor of known stoichiometry (Im et al., 1995; Baumann et al., 2001
, 2002
, 2003
). It was recently shown (Zhou et al., 2003
) that oocyte expression of linked
4 and ß2 nicotinic subunits together with either an
or a ß monomer construct does produce functional nAChRs.
We found that expressing 3ß4 nAChRs from ß4_
3 tandems together with ß4 monomers produced a heterogeneous population of channels, because of incomplete incorporation of the ß subunit from the tandem construct (ßtandem). The cartoons in each panel of Fig. 1 show the three possible receptor assemblies, from full incorporation of all the ßtandem subunits (left) to partial incorporation (middle) to no incorporation (right). In our experiments (Fig. 1, BF), expression of a reporter mutation (star) in different subunits detected incomplete tandem incorporation, because of differences in the numbers of mutations in the channel gate (shown by the numbers under the cartoons). Only a proportion of linked subunit receptors fully incorporated the fusion protein (i.e., are assembled as shown in the cartoons on the left of the panels).
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MATERIALS AND METHODS |
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The mutants in 9' (3L279T and ß4L272T, were L stands for leucine and T for threonine respectively) were created using the QuickChangeTM site-directed mutagenesis kit (Stratagene) and their full-length sequence was verified.
Construction of Tandem Subunit cDNAs
All nine tandem constructs (3_ß4, ß2_
2, ß2_
3, ß2_
4, ß2_
6, ß4_
2, ß4_
3, ß4_
4, and ß4_
6) were made in an identical fashion. First, the coding region of each subunit (
2,
3,
4,
6, ß2, ß4) was amplified by PCR excluding the stop codon. The PCR primers directed to each corresponding start codon included the Kozak consensus sequence (GCCACC) and the EcoRI enzyme restriction site (5'-end PCR-fragment; CTGAATTCGCCACCATG...). The primer directed to the coding sequence upstream of the stop codon included the NotI enzyme restriction site (basically the stop codon is replaced by the NotI restriction site). The resulting DNA fragments were purified and subcloned into the pcDNA3.1/Myc-His version C vector (Invitrogen), using the EcoRI and NotI restriction sites.
A linker DNA fragment was created (based on Im et al., 1995) by hybridization of two complementary oligonucleotides; 5'-GGCCGCTCAGCAACAGCAGCAACAGCAGCAAG-3' and 5'-AATTCTTGCTGCTGTTGCTGCTGTTGCTGAGC-3'. The resulting double-strand DNA linker contains a 5'-end NotI restriction site overhang (underlined) and a 3'-end EcoRI restriction site overhang (underlined), separated by 25 nucleotides (the first nucleotide [bold] is inserted to bring the NotI site [8-cutter] back in the correct reading frame, whereas the next 24 nucleotides code for the eight glutamine amino acids).
The tandem constructs were created using three unique restriction sites; EcoRI (upstream of the start codon of all subunits and the 3'-end of the linker), NotI (downstream of the coding sequence of all subunits and the 5'-end of the linker), and AgeI (between the Myc and His epitope sequences in the pcDNA3.1/Myc-His version C vector). A three way ligation resulted in the following tandem circular plasmid: * [AgeI...His-epitope...stop codon...pcDNA3.1/Myc-His C vector...EcoRI subunit A NotI] * [NotI linker EcoRI] * [EcoRI subunit B NotI...Myc-epitope...AgeI] * (where * represents the ligation sites and [ ] represents purified DNA fragments digested with the restriction sites indicated in bold).
To remove the epitope tags (Myc- and His-) all tandems were subcloned in the corresponding pcDNA3.1 vector, using a unique restriction enzyme site in subunit B. For instance, cutting the tandem upstream of the start codon (of subunit A) and somewhere in subunit B and transferring this fragment in the same position of subunit B, previously cloned in the pcDNA3.1 vector. Finally, all the tandem constructs were also subcloned in the pSP64GL vector. The length of the extracellular linkage of the different tandem constructs differs depending on the extracellular region downstream of TM4 of the first subunit and the length of the signal peptide of the second subunit (Table I).
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The 3_ß4, ß4_
3, ß4_
3L279T, and ß4L272T_
3 tandem constructs cloned in the pSP64GL vector were sequenced fully to check for PCR and/or cloning artifacts. All other tandem constructs were sequenced only at the outer ends to check for cloning artifacts.
Mammalian Cell Culture and Transfection
HEK293 cells were obtained from the American Type Culture Collection (ATCC-CRL-1573). Cells were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with sodium pyruvate (0.11 g/liter), 10% (vol/vol) heat-inactivated fetal bovine serum, 100 U/ml penicillin G, 100 µg/ml streptomycin sulfate, and 2 mM L-glutamine (all from GIBCO BRL) as described previously (Beato et al., 2002). Dishes (10-cm diameter) were plated 24 h before transfection in order to reach around 40% confluence (before transfection). Transfection with LipofectAMINETM (GIBCO BRL) was performed according to the manufacturer's instructions. Briefly, for each dish 24 µl LipofectAMINE and 6 µg cDNA were added to two separate vials each containing serum-free medium (Optimem; GIBCO BRL) to a final volume of 600 µl. The contents of the LipofectAMINE and the cDNA vials were then mixed and incubated for 30 min at room temperature. Finally, 4.8 ml of Optimem medium was added to the mixture and the whole mixture was added dropwise to one dish of cells (washed with 6 ml Optimem and aspirated) and incubated for 40 h at 37°C and 5% CO2 (transfection mixture was replaced with fresh growth medium after 16 h, see above).
Preparation of In-Vitro Capped mRNA and RNA Gel-Electrophoresis
All cDNA/pSP64GL plasmids were linearized immediately downstream of the 3' untranslated ß-globin sequence, and capped cRNA was transcribed using the SP6 Mmessage MmachineTM Kit (Ambion) according to the manufacturer's instructions. For RNA electrophoresis a 1.5% agarose gel was prepared using the 1x gel prep/running buffer (NorthernMax-GlyTM system; Ambion). RNA samples (including the 0.249.5 Kb RNA ladder; GIBCO BRL) were diluted 1:1 with Glyoxal sample loading dye (Ambion) and incubated at 50°C for 30 min before loading. Samples were separated at 5 V/cm for 3 h and RNAs were visualized by UV transillumination and a photograph taken.
Xenopus Oocyte Preparation
Female Xenopus laevis frogs were anesthetized by immersion in neutralized ethyl m-aminobenzoate solution (tricaine, methanesulphonate salt; 0.2% solution wt/vol; Sigma-Aldrich), and killed by decapitation and destruction of the brain and spinal cord (in accordance with Home Office guidelines) before removal of ovarian lobes. Clumps of stage V-VI oocytes were dissected in a sterile modified Barth's solution of composition (in mM): NaCl 88, KCl 1, MgCl2 0.82, CaCl2 0.77, NaHCO3 2.4, Tris-HCl 15, with 50 U/ml penicillin and 50 µg/ml streptomycin, pH 7.4 adjusted with NaOH. The dissected oocytes were treated with collagenase (type IA; Sigma-Aldrich; 65 min at 18°C, 245 collagen digestion U/ml in Barth's solution, 1012 oocytes/ml), rinsed, stored at 4°C overnight, and manually defolliculated the following day before cRNA injection.
Xenopus Oocyte cRNA Injection
For Western blotting, oocytes (10 for each sample) were injected with 46 nl water or 500 ng cRNA (in 46 nl of RNase-free water per oocyte). For two-electrode voltage-clamp recording cRNA was injected at a ratio of 1:1 in order to express 3 + ß4 pair receptors, and at a molar ratio of 2:1 (equals 4:1 cRNA mass) of tandem versus monomer, respectively, in order to express tandem-containing receptors (all in 46 nl of RNase-free water per oocyte). The total amount of cRNA to be injected for each combination was determined empirically with the aim of achieving a maximum ACh-evoked current of 12 µA and was 0.54 ng/oocyte, unless otherwise stated (see Fig. 3 and Table II). Oocytes were incubated for
60 h at 18°C in Barth's solution containing 5% heat-inactivated horse serum (GIBCO BRL; Quick and Lester, 1994
) and then stored at 4°C. Two-electrode voltage-clamp experiments were performed at a room temperature of 1820°C between 2.5 and 5 d from injection.
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Transfected HEK293 cells were (10-cm dish each sample) washed once with Hanks medium (GIBCO BRL) and dislodged by brief exposure to Trypsin (GIBCO BRL). After adding 1x PBS (GIBCO BRL), cell suspension was centrifuged (10 min, 1,000 rpm) and the pellet washed twice with 1 ml PBS. Pellets were homogenized by vigorous pipetting in 1 ml HEK293 lysis buffer (500 mM NaCl, 50 mM NaH2PO4, 1% Triton X-100, 1% protease inhibitor cocktail for mammalian tissues [Sigma-Aldrich], pH 8.0) and rotating at 4°C for 12 h. To pellet cellular debris and DNA the samples were centrifuged at 20,000 g and 4°C for 60 min. A clear 100-µl supernatant sample was taken and proteins precipitated by adding 400 µl methanol, 100 µl chloroform, and 300 µl water, respectively, and centrifugation at 20,000 g for 3 min at 4°C. Subsequently, most of the top layer was removed (protein is at the interface) and 100 µl methanol was added followed by centrifugation at 20,000 g for 5 min at 4°C. Finally, the protein pellets were resuspended in 50 µl Laemmli sample buffer (Bio-Rad Laboratories) containing 5% ß-mercapto-ethanol (Bio-Rad Laboratories).
Western Blots
Both HEK293 and oocyte protein samples were incubated at 75°C for 8 min just before loading (20 µl each sample) on a 8% Tris-Glycine polyacrylamide gel containing 2% SDS together with the SeeBlue® prestained protein standard (Invitrogen). After PAGE-SDS the proteins were transferred to nitrocellulose membrane (0.2 µm, protran BA83; Schleicher-Schuell). The blots were probed with rabbit antiserum to 3 or ß4 (diluted 1:200 from 5-ml stock solution; Research & Diagnostic antibodies, WR-5611 [
3] and WR-5656 [ß4]) followed by HRP-labeled goat antirabbit IgG (diluted 1:10,000, 10 µg/ml stock solution from supersignal® west femto chemiluminescence substrate kit; Pierce Chemical Co.). After washing, blots were visualized using the supersignal® west femto chemiluminescence substrate kit (Pierce Chemical Co.) and exposure to biomax light films (Kodak).
Two-electrode Voltage-clamp Recording
Oocytes, held in a 0.2 ml bath, were perfused at 4.5 ml/min with modified Ringer solution (in mM): NaCl 150, KCl 2.8, HEPES 10, MgCl2 2, atropine sulfate 0.5 µM (Sigma-Aldrich), pH 7.2 adjusted with NaOH and voltage clamped at 70 mV, using the two-electrode clamp mode of an Axoclamp-2B amplifier (Axon Instruments, Inc.). Electrodes were pulled from Clark borosilicate glass GC150TF (Warner Instrument Corporation) and filled with 3 M KCl. The electrode resistance was 0.51 M on the current-passing side. Experiments were terminated if the total holding current exceeded 2 µA, in order to reduce the effect of series resistance errors. We chose a nominally calcium-free solution in order to minimize the contribution of calcium-gated chloride conductance; this is endogenous to the Xenopus oocyte and may be activated by calcium entry through the neuronal nicotinic channels (Sands et al., 1993
).
The agonist solution (acetylcholine chloride [Sigma-Aldrich], freshly prepared from frozen stock aliquots) was applied via the bath perfusion for a period sufficient to obtain a stable plateau response (at low concentrations) or the beginning of a sag after a peak (at the higher concentrations). The resulting inward current was recorded on a flat bed chart recorder (Kipp & Zonen) for later analysis. An interval of 5 min was allowed between ACh applications, as this was found to be sufficient to ensure reproducible responses. A descending dose protocol was used. To compensate for possible decreases in agonist sensitivity during the experiment, a standard concentration of ACh (approximately EC20 for the particular combination used) was applied every third response for concentration-response curves. The experiment was started only after checking that this standard concentration gave reproducible responses. All the data shown in the study are compensated for the response rundown (Boorman et al., 2000). The average rundown in response to the standard concentrations for the different receptor constructs were:
3 + ß4, 40 ± 3%; ß4_
3 + ß4, 39 ± 11%; ß4_
3 + ß4LT, 68 ± 5%; ß4LT_
3 + ß4, 45 ± 4%; ß4_
3LT + ß4, 57 ± 4% and ß4LT_
3 + ß4LT, 79 ± 3%.
To reassure ourselves that the lack of functional expression observed for some subunit combinations was true and not a false negative due to oocyte health or expression problems contingent to a given batch, oocyte data were obtained from a minimum of two separate oocyte batches for each combination. Each batch was from a different frog, processed on different experimental weeks: in every experimental batch at least one "control" highly expressing subunit combination was injected to check for expression efficiency.
Curve Fitting
Doseresponse curves were fitted with the Hill equation:
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Fitting was done in stages, as follows. Each doseresponse curve was fitted separately, individual responses being equally weighted, in order to obtain estimates for Imax, EC50, and nH (see RESULTS). The doseresponse curves shown in Figs. 2 and 57 were obtained by normalizing each datapoint to the fitted maximum response in that oocyte before pooling and fitting the pooled data with the Hill equation (with weight given by the reciprocal of their variance). Parameter estimates were similar to those obtained by fitting each curve separately.
When two-components were detected in the concentration-response curve (see Fig. 7), free fits of the individual dose response curves were poorly defined because of the large number of parameters fitted. Good fits were obtained when all the concentration-response curves for this combination were fitted simultaneously with EC50 and nH values for the two components constrained to be equal across oocytes, while the proportion of the current in the first component was allowed to vary from one oocyte to the other.
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RESULTS |
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Therefore, we expressed either of the linked constructs 3_ß4 or ß4_
3 together with ß4 (in a molar ratio of 2:1 tandem:monomer). After injection of the
3_ß4 + ß4 cRNA combination, there was no functional expression, i.e., no response to 1 mM ACh (n = 5). In contrast with that, the linked construct with the opposite orientation (ß4_
3) expressed efficiently with ß4, giving an average maximum inward current to ACh in oocytes of 2.6 µA (n = 10, Table II, Fig. 2, 2 ng ß4_
3:0.5 ng ß4 cRNA injected), whereas injection of ß4_
3 tandem alone gave no expression (1 mM ACh, n = 6 oocytes from two different batches, 2 ng of cRNA, see Table II), suggesting proper incorporation of the tandem constructs.
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Linked subunits with opposite orientation differ in two points, namely in the position of the linker with respect to the agonist binding site domains (Zhou et al., 2003) and in the length of the effective linker (i.e., the introduced linker together with the COOH terminus extracellular domain of the leading subunit and the signal peptide of the second subunit), which is shorter in
3_ß4 than in ß4_
3 because of the differences in the COOH-terminal end/signal peptide of the two subunits (Table I). Either of these two factors may have prevented functional expression when the tandem orientation is
_ß. Another possibility is that the
3_ß4 orientation does not allow the assembly of the receptor subunits in the correct order (if assembly is similar to that of muscle nAChRs; see Green, 1999
).
Expression of other Tandem Constructs Alone or Together with ß2 or ß4
We proceeded to test which other ß_ + ß combinations gave functional nAChR, bearing in mind that the subunits known to form "pair" nAChRs in oocytes are
2,
3, or
4 together with either ß2 or ß4. In all oocytes injected with linked constructs of the form ß4_
+ ß4, ACh application gave rise to large inward currents, indicating efficient production of nAChRs (maximum currents in response to 1 mM ACh are shown in Fig. 3, gray bars). On the other hand, of all the ß2-containing tandems, the only combination that produced significant currents was ß2_
4 + ß2 (Fig. 3). No functional response was observed when subunit dimers containing
6 (ß2_
6 + ß2 and ß4_
6 + ß4) were expressed (n = 6 and 5, respectively; not depicted).
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We next checked that the tandem cRNA used in the transfections was pure and had appropriate molecular weight. This is shown by the gel in Fig. 4 A, where cRNAs for the 3 or ß4 monomers (lanes 1 and 2) and those for the
3_ß4 and ß4_
3 tandem constructs (lanes 3 and 4) migrate as single bands at the molecular weights expected (
1.8 and 3.4 Kb for single subunits and tandems, respectively). We sought additional confirmation that the actual fusion protein formed by expression of the tandem construct ß4_
3 does not undergo proteolysis by analyzing the protein formed by SDS-PAGE fractionation, Western blotting, and detection by antiserum raised against the ß4 subunit. This antibody detected a single band at the appropriate molecular weight (
56 kD) in proteins solubilized from oocytes or HEK-293 cells transfected with the ß4 monomer subunit alone (Fig. 4, B and C). However, there was no signal in proteins from oocytes or cells transfected with the tandem construct ß4_
3.
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In ß4_3 + ß4 nAChR Two Copies of
3 and Three Copies of ß4 Participate in Channel Gating
Another requisite for the validation of the tandem construct expression technique is that the receptor formed from linked subunits should have the same stoichiometry as receptors expressed from monomeric constructs. In the case of oocyte-expressed 3ß4 nAChRs, this means two copies of
3 and three of ß4 (Boorman et al., 2000
). A simple way of checking stoichiometry is to apply to linked-subunit receptors the reporter mutation approach that we have previously exploited for the
3ß4 and
3ß4ß3 combinations (Boorman et al., 2000
). Mutating the 9' hydrophobic amino acid (Leu or Val) in the pore-lining second transmembrane domain of a nicotinic type subunit to a hydrophilic residue (Thr or Ser) makes the resulting receptor more sensitive to agonists (Revah et al., 1991
), probably by destabilizing the closed state. The extent to which the agonist EC50 is reduced is approximately proportional to the number of mutant subunits in the pentamer (Labarca et al., 1995
; Chang et al., 1996
; Chang and Weiss, 1999
).
Fig. 5 shows the effect of inserting a 9' LT mutation in either all the ß4 subunits (filled triangles, leftmost curve) or all the 3 subunits (filled squares, middle curve; the dashed curve shows for reference the position of the wild-type tandem receptor) expressed in the linked ß4_
3 + ß4 nAChR (see Fig. 2). The mutation produces a greater leftward shift in the ACh doseresponse curve if it is expressed in the ß4 subunit, reducing the wild-type EC50 of 122 ± 8 µM to 0.68 ± 0.02 µM (ß4LT_
3 + ß4LT, n = 7). A smaller effect is seen when
3 subunits are mutated, with a decrease in the EC50 to 3.81 ± 0.18 µM (ß4_
3LT + ß4, n = 8). This indicates clearly that more ß than
subunits contribute to the channel gate in the linked subunit receptor. This pattern is identical to that seen in
3 + ß4 nAChRs, where mutating ß4 or
3 produced a 292-fold or a 37-fold reduction in EC50, respectively (Boorman et al., 2000
). It is therefore reasonable to conclude that linked-subunit nAChR contain three ß4 and two
3 subunits in the channel gating domain.
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This hypothesis was confirmed by further experiments in which the ß4_3LT tandem construct was expressed together with ß4LT, a combination that should in principle give rise to a nAChR with three mutation copies (Fig. 1 F, left). However, poor survival was observed for oocytes injected with this combination over the 2 d of incubation; the few surviving oocytes (25%) had high holding current, which was reduced by 46 ± 6% (n = 3) by application of a high concentration of the nicotinic channel blocker trimetaphan (10 µM). Both the high holding current and the poor oocyte health are likely to be due to spontaneous opening of nAChRs and were similar to observation of receptors with 5 mutations (
3LT + ß4LT; not depicted). Such phenomena were not detectable for nAChRs with three mutation copies or less (with or without linked subunits) and suggest again that the monomer construct contributes more than one copy of the ß4 subunit to the linked subunit receptor complex (Fig. 1 F, right).
It is therefore clear that the concatemer technique fails the ultimate test for providing a method for producing nAChR of defined subunit composition, as ß4_3 + ß4 receptors do not contain two copies of ß4_
3 and one of ß4. Simply checking the ACh sensitivity of the receptor was not sufficient to detect this receptor heterogeneity.
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DISCUSSION |
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Using a short linker, we obtained efficient functional expression for several pair-type combinations (2ß4,
3ß4,
4ß4, and
4ß2).
3ß4-type receptors were chosen for further characterization, as the tandem construct for this combination (ß4_
3) gave no nicotinic currents if expressed alone, but showed robust expression if expressed together with ß4, suggesting that the resulting receptor population may be constituted entirely by pentamers with two copies of
and three of ß. These ß4_
3 + ß4 tandem nAChRs were very similar to their nontandem equivalent,
3 + ß4 nAChRs, in that they had identical doseresponse curves to ACh and responded in the same way to the introduction of a reporter mutation in the TM2 domain of either all the
or all the ß subunits expressed. This indicated that the channel function was not grossly disrupted by the linker in either binding or gating and that the channel gate contained the same subunit types for both wild-type and linked subunit receptors, namely two
and three ß subunits. Nevertheless, mutating only a subset of the ß subunit constructs (i.e., only ß4tandem or ß4monomer) revealed heterogeneity in the ACh sensitivity of linked-subunit receptors. This was probably due to channels containing variable numbers of mutations (i.e., ß subunits from monomers or tandems) and ultimately to incomplete incorporation of the ß4tandem subunit. This means that a substantial fraction of the pentamers may incorporate only the COOH terminus
of the fusion protein into the channel.
This hypothesis is illustrated in Fig. 1, AC, which shows that experiments in which all or all ß subunits are mutated do not give any information on the origin of these subunits. Fig. 1 D shows how a mix of the different types of pentamers (i.e., one, two, or three ß subunits from monomer) explains well the presence and properties of more than one component in the ACh doseresponse curve when ß4monomer was mutated (Fig. 7). These components are likely to represent channels that contain either one or three mutation copies. Because of the limited sensitivity of fitting several components to a doseresponse curve, we cannot exclude or quantify the presence of an intermediate fraction of receptors containing two copies of the ßmonomer.
In the light of this hypothesis, it is hard to account fully for the results of the complementary experiment in which ß4tandem was mutated. The ACh sensitivity of these receptors was only slightly greater than that of wild-type receptors, suggesting that most linked subunit receptors contained only ß subunits from the monomer. This is in contrast with the estimate that the equivalent receptors when ßmonomer carried the mutation (high-sensitivity component in Fig. 7) accounted for 60% of current. However, it is worth noting that in the latter experiment, there was great variability between oocytes in the proportion of high-sensitivity current and that in 3 out of 13 oocytes this was the only component detectable.
Can Alternate Stoichiometries Account for Our Results?
It was recently shown (Zhou et al., 2003) that tandem constructs of
4ß2 subunits give functional expression in the absence of a monomer construct, a situation analogous to that described for the expression of P2X receptors (which are trimeric) from tandem constructs alone (Stoop et al., 1999
; Nicke et al., 2003
). Nicotinic
4ß2 tandem constructs form dipentamers of receptors, half of which contain two
subunits and half three
(in accord with the possibility that this combination may exist in two different subunit stoichiometries; Nelson et al., 2003
). While it was reported that these dipentamers disappeared when expressed with monomer constructs, we must consider the possibility that the receptors formed by our tandem constructs include a similar dipentamer alone, or together with a correctly assembled pentamer (i.e., one containing one ß4monomer). It is unlikely that this possibility accounts for our findings for several reasons, first of all because the ß4_
3 tandem alone does not produce functional receptors. If we assume that a dipentamer does nevertheless form, this would predict that receptors with all
or all ß subunit mutated would contain two and a half mutation copies on average (as half would have two and half would have three). In turn this would suggest that, if mutations are equivalent, these different mutant combinations should have the same sensitivity to ACh (i.e., the two curves in Fig. 5 should coincide). Finally, receptors in the dipentamer are formed exclusively by tandem constructs. In the expression of receptors in which only the monomer is mutated this would predict a significant wild-type component (which we did not detect).
Similarly, it is unlikely that the results of mutating ß4monomer can be explained by populations of receptors with different numbers of subunits. If
3ß4 nAChRs with three
subunits are present, they must represent a small proportion of the total, given the clear-cut results of mutating all
or all ß subunits. Furthermore, a three
subunit receptor could in principle form from the expression of tandem constructs of opposite orientation or of ß4_
3 +
3, neither of which produced functional receptors.
Another possibility is that linked subunit receptors assemble correctly, but that the characteristics of the hydrophilic 9' mutation in TM2 are altered in a way that curtails their usefulness in establishing subunit incorporation or stoichiometry. It could be that, even though the wild-type ACh sensitivity is unchanged, the conformational constraints introduced by linking the subunits distort the channel to such an extent that the magnitude of the effect of the reporter mutation becomes dependent on the subunit that carries it and on the number of mutations already in the pore, rendering the results uninterpretable. It is hard to see how that could explain the two components in the doseresponse curve for the receptor from mutant ß4monomer. At any rate, this hypothesis entails that the mutation has different properties in linked subunit receptors versus 3 + ß4 receptors. That in itself would mean that the tandem technique has failed in reproducing this channel.
Expression of Concatenated Subunits in Other Channel Superfamilies
The strategy of linking subunits has been exploited in practically every channel family with favorable subunit topology (i.e., both NH2 and COOH termini on the same side of the membrane), yielding a variety of useful data (for review see Nicke et al., 2003). Inconsistencies have emerged, notably for K+ channels (Liman et al., 1992
; McCormack et al., 1992
; Hurst et al., 1995
; Silverman et al., 1996
) and for P2X receptors (Stoop et al., 1999
; Nicke et al., 2003
). Additionally, early results on the stoichiometry of cyclic nucleotidegated channels obtained by linking subunits are contradicted by new studies (Zimmerman, 2002
). Steric hindrance, leading to the incomplete assembly of concatenated subunits into a channel and/or to the formation of higher-order complexes of receptors was invoked to explain such results. Note that in the cartoon in Fig. 1, our working hypothesis, based on similar conjectures, represents unassembled ß4tandem subunits as trailing outside the receptor for simplicity, although it cannot be excluded that they are cleaved before exit from the endoplasmic reticulum, as suggested for P2X1 receptors (Nicke et al., 2003
). An interesting feature is that incomplete dimer incorporation in our case results in the COOH-terminal subunit of the dimer participating to the channel. This is in contrast with the common observation that the NH2-terminal subunit in a multimer incorporates most efficiently, and strengthens our conjecture that premature termination of translation or 3' degradation of cRNA does not take place to produce truncated dimers.
In conclusion, our data show that the incomplete incorporation of the linked subunits and the consequent heterogeneity of the assembled receptor were not detected by a simple doseresponse curve, but required extensive functional testing aided by reporter mutations. Clearly, the ultimate failure of our particular version of the linked subunit strategy does not exclude that it may be possible to engineer a different linker to produce homogeneous defined composition receptors in which the linked subunits incorporate efficiently. The difficulty of this task probably lies in balancing opposing demands on the linker, as a long flexible sequence will allow the formation of functional dimers of different pentamers, whereas a short linker would better constrain stoichiometry but may damage incorporation efficiency of the fusion protein. Nevertheless, our data clearly set out a minimum range of tests for a linked subunit receptor to be deemed to have successfully reproduced the characteristics of the unlinked receptor.
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ACKNOWLEDGMENTS |
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Angus C. Nairn served as editor.
Submitted: 13 February 2004
Accepted: 9 April 2004
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REFERENCES |
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