Heteromeric Neuronal Nicotinic Acetylcholine Receptors with Mutant β Subunits Acquire Sensitivity to α 7-Selective Positive Allosteric Modulators

Homomeric 7 nicotinic acetylcholine receptors (nAChR) have an intrinsically low probability of opening that can be overcome by 7-selective positive allosteric modulators (PAMs), which bind at a site involving the second transmembrane domain (TM2). Mutation of a methionine that is unique to 7 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 4 and 2, which are the nAChR subunits that are most abundant in the brain. Receptors containing these mutations were found to be strongly potentiated by the 7 PAM 3 ,4,5,9 -tetrahydro-4-(1-naphthalenyl)-3 -cyclopentan[ ]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 2 subunit was necessary and sufficient for TQS sensitivity. The primary effect of the mutation in the 4 subunit was to reduce responses to acetylcholine applied alone. Sensitivity to TQS required only a single mutant subunit, regardless of the position of the mutant subunit within the pentameric complex. Similar results were obtained when 2L15'M was coexpressed with 2 or 3 and when the L15'M mutation was placed in 4 and coexpressed with 2, 3, or 4. Functional receptors were not observed when 1L15'M subunits were coexpressed with other muscle nAChR subunits. The unique structure-activity relationship of PAMs and the 4 2L15'M receptor compared with 7 and the availability of high-resolution 4 2 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 α7 subunits but are insensitive to PAMs, which greatly increase the open probability of α7 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 α7. Specifically, the mutation of the TM2 15' leucine to methionine in subunits reduces heteromeric receptor channel activation, while the same mutation in neuronal subunits allows heteromeric receptors to respond to select 7 PAMs. The results indicate a key role for this residue in the functional differences in the two main classes of neuronal nAChRs.