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Tandem constructs are increasingly being used to restrict the composition of recombinant multimeric channels. It is therefore important to assess not only whether such approaches give functional channels, but also whether such channels completely incorporate the subunit tandems. We have addressed this question for neuronal nicotinic acetylcholine receptors, using a channel mutation as a reporter for subunit incorporation. We prepared tandem constructs of nicotinic receptors by linking alpha (alpha2-alpha4, alpha6) and beta (beta2, beta4) subunits by a short linker of eight glutamine residues. Robust functional expression in oocytes was observed for several tandems (beta4_alpha2, beta4_alpha3, beta4_alpha4, and beta2_alpha4) when coexpressed with the corresponding beta monomer subunit. All tandems expressed when injected alone, except for beta4_alpha3, which produced functional channels only together with beta4 monomer and was chosen for further characterization. These channels produced from beta4_alpha3 tandem constructs plus beta4 monomer were identical with receptors expressed from monomer alpha3 and beta4 constructs in acetylcholine sensitivity and in the number of alpha and beta subunits incorporated in the channel gate. However, separately mutating the beta subunit in either the monomer or the tandem revealed that tandem-expressed channels are heterogeneous. Only a proportion of these channels contained as expected two copies of beta subunits from the tandem and one from the beta monomer construct, whereas the rest incorporated two or three beta monomers. Such inaccuracies in concatameric receptor assembly would not have been apparent with a standard functional characterization of the receptor. Extensive validation is needed for tandem-expressed receptors in the nicotinic superfamily.
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15148328
???displayArticle.pmcLink???PMC2234567 ???displayArticle.link???J Gen Physiol ???displayArticle.grants???[+]
Figure 1. . Diagrammatic representation of different nAChR assemblies which may be formed by complete (left) or partial (center and right) incorporation of linked constructs into the receptor. B–F show the effects of introducing a reporter mutation (a Thr in the 9′ position of the second transmembrane domain) in different subunits; references are given to the figures that show the results with each combination. The number under each cartoon shows the number of mutation copies expected to be in the gate for each receptor assembly. Note that no heterogeneity in the number of mutation copies is predicted if all α, all β, or no subunits are mutated (A–C) in accord with the experiments in Figs. 2 and 5. Mutating β only in the monomer construct or only in the tandem construct (D and E) can detect the different receptor forms (see the experiments in Figs. 7 and 6, respectively). If receptors are expressed in which both α and βmonomer (F) bear the mutation, some receptors will bear five copies of the mutation (see results).
Figure 2. . ACh concentration-response curves of α3β4 nAChR expressed in oocytes from monomer or tandem constructs are indistinguishable. (A) Traces are inward currents recorded at a holding potential of −70 mV in response to bath-applied ACh. (B) ACh concentration-response curves from experiments such as the ones shown in A, performed in oocytes injected with either α3 and β4 monomer cRNAs (filled circles, n = 9) or β4_α3 tandem together with β4 monomer cRNAs (open circles, n = 4). Full sets of peak responses to ACh from each oocyte were fitted with the Hill equation and normalised to the fitted maximum response before pooling (see materials and methods). The curves shown are the results of fitting the pooled data.
Figure 3. . Some tandem constructs form functional channels when expressed alone in Xenopus oocytes. Tandem constructs were expressed with or without monomer construct of the same β subunit they contain (β2 or β4; 2 ng/μl tandem to 0.5 ng/μl monomer; 46 nl injected/oocyte). The four tandem constructs shown here were the only ones to produce functional receptors when expressed with the corresponding β monomer (n = 5–10). Note that three out of these four tandem constructs produced inward currents to 1 mM ACh also when expressed alone (2 ng/μl injected, n = 4–6). Only the β4_α3 tandem construct failed to produce any inward currents (n = 6) when expressed alone.
Figure 4. . cRNA gel-electrophoresis (A) and Western blots of expressed proteins in oocytes (B) and HEK293 cells (C). Approximately 1 μg of α3 (1), β4 (2), α3_β4 tandem (3), and β4_α3 tandem (4) besides the RNA ladder (M) were separated on a 1.5% agarose-gel (A). The Western blot in B was obtained from oocytes injected with water only (MQ), β4 only, or β4_α3 tandem (T) and the Western blot in C from HEK293 cells transfected with no DNA (ND), α3 only, β4 only, or β4_α3 tandem (T). Detection by β4 antibody and visualization by chemoluminescence. Bands for the β4 subunit were detected at the expected size of 56 kD for both blots after 30-s exposure. No breakdown products were observed for the tandem constructs in either blots, even for longer exposures up to 1 h. Note that the tandem fusion protein (predicted size of 115 kD) was not detected by the β4 antibody used (see text).
Figure 5. . The effects of inserting a L9′T reporter mutation into all the α or all the β subunits in tandem construct receptors. (A) Examples of inward currents elicited by bath-applied ACh in oocytes expressing β4LT_α3 + β4LT (top) or β4_α3LT + β4 (bottom). (B) Concentration-response curves from oocytes injected with β4LT_α3 + β4LT cRNAs (filled triangles, n = 7) or β4_α3LT + β4 cRNAs (filled squares, n = 8). For details of the fitting see materials and methods and the legend to Fig. 2. The concentration-response curve for the β4_α3 + β4 wild-type nAChR (from Fig. 2 B) is shown for reference (dotted line). The EC50 shifts produced by the mutation are similar to those observed in α3 + β4 receptors and suggest that the channel gate is made up of two α and three β subunits both in linked-subunit and in monomer construct receptors.
Figure 6. . Low ACh sensitivity of linked subunit receptors carrying the L9′T reporter mutation in β4tandem only. (A) Examples of inward currents elicited by bath-applied ACh in oocytes expressing β4LT_α3 + β4. (B) Data from oocytes injected with β4LT_α3 + β4 cRNAs (filled squares, n = 7). Fits as in Fig. 2. The concentration-response curve for β4_α3 + β4 wild-type (see Fig. 2 B) is shown for reference (dotted line), together with the concentration-response curve for the β4_α3LT + β4 combination (from Fig. 4 B, dashed line): the latter shows the EC50 shift expected with the incorporation of two L9′T mutations.
Figure 7. . Inserting the L9′T reporter mutation in the β4monomer subunit of linked subunit nAChRs reveals multiple receptor populations. (A) Examples of inward currents elicited by bath-applied ACh in oocytes expressing β4_α3 + β4LT. (B) Concentration-response curve for oocytes injected with β4_α3 + β4LT cRNAs (filled circles, n = 4). Data were normalized and pooled as described in materials and methods and in the legend to Fig. 2. In this case the initial fit was a simultaneous fit of a two-component Hill equation to each dose–response curve, with the constraint of equal EC50 and Hill slope across oocytes (the proportion of the first component was allowed to vary). The three dotted curves shown for reference are, respectively (right to left), the concentration-response curve for receptors with no mutations (β4_α3 + β4), two mutation copies (β4_α3LT + β4), or three mutation copies (β4LT_α3 + β4LT). The dashed curve shows the concentration-response curve expected for complete tandem incorporation as depicted in the cartoon (i.e., one mutation copy).
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