Stokes_2019_J.Pharmacol.Exp.Ther_370_252

Homomeric alpha7 nicotinic acetylcholine receptors (nAChR) have an intrinsically low probability of opening that can be overcome by alpha7-selective positive allosteric modulators (PAMs), which bind at a site involving the second transmembrane domain (TM2). Mutation of a methionine that is unique to alpha7 at the 15' position of TM2 to leucine, the residue in most other nAChR subunits, largely eliminates the activity of such PAMs. We tested the effect of the reverse mutation (L15'M) in heteromeric nAChR receptors containing alpha4 and beta2, which are the nAChR subunits that are most abundant in the brain. Receptors containing these mutations were found to be strongly potentiated by the alpha7 PAM 3a,4,5,9b-tetrahydro-4-(1-naphthalenyl)-3H-cyclopentan[c]quinoline-8-sulfonamide (TQS) but insensitive to the alternative PAM 1-(5-chloro-2,4-dimethoxyphenyl)-3-(5-methylisoxazol-3-yl)-urea. The presence of the mutation in the beta2 subunit was necessary and sufficient for TQS sensitivity. The primary effect of the mutation in the alpha4 subunit was to reduce responses to acetylcholine applied alone. Sensitivity to TQS required only a single mutant beta subunit, regardless of the position of the mutant beta subunit within the pentameric complex. Similar results were obtained when beta2L15'M was coexpressed with alpha2 or alpha3 and when the L15'M mutation was placed in beta4 and coexpressed with alpha2, alpha3, or alpha4. Functional receptors were not observed when beta1L15'M subunits were coexpressed with other muscle nAChR subunits. The unique structure-activity relationship of PAMs and the alpha4beta2L15'M receptor compared with alpha7 and the availability of high-resolution alpha4beta2 structures may provide new insights into the fundamental mechanisms of nAChR allosteric potentiation. SIGNIFICANCE STATEMENT: Heteromeric neuronal nAChRs have a relatively high initial probability of channel activation compared to receptors that are homomers of alpha7 subunits but are insensitive to PAMs, which greatly increase the open probability of alpha7 receptors. These features of heteromeric nAChR can be reversed by mutation of a single residue present in all neuronal heteromeric nAChR subunits to the sequence found in alpha7. Specifically, the mutation of the TM2 15' leucine to methionine in alpha subunits reduces heteromeric receptor channel activation, while the same mutation in neuronal beta subunits allows heteromeric receptors to respond to select alpha7 PAMs. The results indicate a key role for this residue in the functional differences in the two main classes of neuronal nAChRs.