Groot-Kormelink PJ et al. (2004), Incomplete incorporation of tandem subunits in ...

XB-ART-3524

J Gen Physiol 2004 Jun 01;1236:697-708. doi: 10.1085/jgp.200409042.

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Incomplete incorporation of tandem subunits in recombinant neuronal nicotinic receptors.

Groot-Kormelink PJ , Broadbent SD , Boorman JP , Sivilotti LG .

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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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Species referenced: Xenopus laevis
Genes referenced: chrna4

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	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. BâF show the effects of introducing a reporter mutation (a Thr in the 9â² 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 Î±, all Î², or no subunits are mutated (AâC) in accord with the experiments in Figs. 2 and 5. Mutating Î² 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 Î± and Î²monomer (F) bear the mutation, some receptors will bear five copies of the mutation (see results).
	Figure 2. . ACh concentration-response curves of Î±3Î²4 nAChR expressed in oocytes from monomer or tandem constructs are indistinguishable. (A) Traces are inward currents recorded at a holding potential of â70 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 Î±3 and Î²4 monomer cRNAs (filled circles, n = 9) or Î²4_Î±3 tandem together with Î²4 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 Î² subunit they contain (Î²2 or Î²4; 2 ng/Î¼l tandem to 0.5 ng/Î¼l monomer; 46 nl injected/oocyte). The four tandem constructs shown here were the only ones to produce functional receptors when expressed with the corresponding Î² monomer (n = 5â10). Note that three out of these four tandem constructs produced inward currents to 1 mM ACh also when expressed alone (2 ng/Î¼l injected, n = 4â6). Only the Î²4_Î±3 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 Î¼g of Î±3 (1), Î²4 (2), Î±3_Î²4 tandem (3), and Î²4_Î±3 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), Î²4 only, or Î²4_Î±3 tandem (T) and the Western blot in C from HEK293 cells transfected with no DNA (ND), Î±3 only, Î²4 only, or Î²4_Î±3 tandem (T). Detection by Î²4 antibody and visualization by chemoluminescence. Bands for the Î²4 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 Î²4 antibody used (see text).
	Figure 5. . The effects of inserting a L9â²T reporter mutation into all the Î± or all the Î² subunits in tandem construct receptors. (A) Examples of inward currents elicited by bath-applied ACh in oocytes expressing Î²4LT_Î±3 + Î²4LT (top) or Î²4_Î±3LT + Î²4 (bottom). (B) Concentration-response curves from oocytes injected with Î²4LT_Î±3 + Î²4LT cRNAs (filled triangles, n = 7) or Î²4_Î±3LT + Î²4 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 Î²4_Î±3 + Î²4 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 Î±3 + Î²4 receptors and suggest that the channel gate is made up of two Î± and three Î² subunits both in linked-subunit and in monomer construct receptors.
	Figure 6. . Low ACh sensitivity of linked subunit receptors carrying the L9â²T reporter mutation in Î²4tandem only. (A) Examples of inward currents elicited by bath-applied ACh in oocytes expressing Î²4LT_Î±3 + Î²4. (B) Data from oocytes injected with Î²4LT_Î±3 + Î²4 cRNAs (filled squares, n = 7). Fits as in Fig. 2. The concentration-response curve for Î²4_Î±3 + Î²4 wild-type (see Fig. 2 B) is shown for reference (dotted line), together with the concentration-response curve for the Î²4_Î±3LT + Î²4 combination (from Fig. 4 B, dashed line): the latter shows the EC50 shift expected with the incorporation of two L9â²T mutations.
	Figure 7. . Inserting the L9â²T reporter mutation in the Î²4monomer subunit of linked subunit nAChRs reveals multiple receptor populations. (A) Examples of inward currents elicited by bath-applied ACh in oocytes expressing Î²4_Î±3 + Î²4LT. (B) Concentration-response curve for oocytes injected with Î²4_Î±3 + Î²4LT 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 doseâresponse 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 (Î²4_Î±3 + Î²4), two mutation copies (Î²4_Î±3LT + Î²4), or three mutation copies (Î²4LT_Î±3 + Î²4LT). 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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