Species Selectivity of a Nicotinic Acetylcholine Receptor Agonist Is Conferred by Two Adjacent Extracellular β4 Amino Acids that Are Implicated in the Coupling of Binding to Channel Gating

Abstract

5-(Trifluoromethyl)-6-(1-methyl-azepan-4-yl)methyl-1H-quinolin-2-one (TMAQ) is a novel nicotinic acetylcholine receptor (nAChR) agonist with strong selectivity for beta4-containing receptors. TMAQ also exhibits remarkable species selectivity, being a potent agonist of nAChRs containing the human beta4 subunit but having no detectable agonist activity on nAChRs containing the rat beta4 subunit. With the aim of identifying subunit domains and individual amino acids, which contribute to the species selectivity of TMAQ, a series of chimeric and mutated beta4 subunits has been constructed. Recombinant receptors containing wild-type, chimeric, or mutated beta4 subunits have been examined by radioligand binding, intracellular calcium assays, and electrophysiological recording. Two adjacent amino acids located within the extracellular loop D domain of the beta4 subunit (amino acids 55 and 56) have been identified as playing a critical role in determining the agonist potency of TMAQ. Mutagenesis of these two residues within the rat beta4 subunit to the corresponding amino acids in the human beta4 subunit (S55N and I56V mutations) confers sensitivity to TMAQ. The converse mutations in the human beta4 subunit (N55S and V56I) largely abolish sensitivity to TMAQ. In contrast, these mutations have little or no effect on sensitivity to the nonselective nicotinic agonist epibatidine. Despite acting as a potent agonist of human beta4-containing nAChRs, TMAQ acts as an antagonist of rat beta4-containing receptors. Our experimental data, together with homology models of the rat and human alpha3beta4 nAChRs, suggest that amino acids 55 and 56 may be involved in the coupling of agonist binding and channel gating.

Agonist activation of α7 nicotinic acetylcholine receptors via an allosteric transmembrane site

“…Xenopus laevis oocytes were isolated and defolliculated, as described previously (52). Heterologous expression was achieved by injection of either cRNA (6-12 ng) into oocyte cytoplasm in the case of wild-type and mutated α7, or plasmid cDNA constructs (10-30 ng) into oocyte nuclei in the case of 5-HT3A and α7/5-HT3A subunit chimeras.…”

“…Oocytes were injected in a volume of 32.2 nL using a Drummond variable volume microinjector. Two electrode voltage-clamp recordings were performed essentially as described previously (52).…”

Potentiation of α7 nicotinic acetylcholine receptors via an allosteric transmembrane site

“…42. For ␣7 and 5-HT3A, oocyte nuclei were injected with cDNAs cloned downstream from a CMV promoter in plasmid expression vectors (42). For ␣8, cytoplasmic injection of cRNA was used to enhance levels of functional expression and cRNA was synthesised using mMESSAGE mMACHINE T7 transcription kit (Ambion).…”

“…42. For ␣7 and 5-HT3A, oocyte nuclei were injected with cDNAs cloned downstream from a CMV promoter in plasmid expression vectors (42).…”

A review of experimental techniques used for the heterologous expression of nicotinic acetylcholine receptors

“…Other studies involving the use of subunit chimeras have helped to investigate receptor properties such as agonist sensitivity [188][189][190][191][192][193][194][195], antagonist sensitivity [190,192,[196][197][198], modulation by allosteric modulators [199,200], desensitization [188], inactivation [201] and channel gating [202][203][204]. M a n u s c r i p t 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 14 Fusion proteins, in which nAChR subunits are linked to proteins such as GFP (green fluorescent protein) have been useful in detecting recombinant subunits expressed in either cells or tissues …”