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J Pharmacol Exp Ther. 2019 Jun 7. pii: jpet.119.259499. doi: 10.1124/jpet.119.259499. [Epub ahead of print]

Heteromeric neuronal nicotinic acetylcholine receptors with mutant beta subunits acquire sensitivity to α7-selective positive allosteric modulators.

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University of Florida.
Northeastern University.
UT Southwestern Medical Center.
University of Texas Southwestern Medical Center.
University of Florida;


Homomeric α7 nicotinic acetylcholine receptors (nAChR) have an intrinsically low probability of opening that can be overcome by α7-selective positive allosteric modulators (PAMs), which bind at a site involving the second transmembrane domain (TM2). Mutation of a methionine, that is unique to α7 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 α4 and β2, the nAChR subunits that are most abundant in brain. Receptors containing these mutations were found to be strongly potentiated by the α7 PAM TQS but insensitive to the alternative PAM PNU-120596. The presence of the mutation in the β2 subunit was necessary and sufficient for TQS sensitivity. The primary effect of the mutation in the α4 subunit was to reduce responses to ACh applied alone. Sensitivity to TQS required only a single mutant β subunit, regardless of the position of the mutant β subunit within the pentameric complex. Similar results were obtained when β2L15'M was co-expressed with the alternative α subunits (α2 or α3) and when the L15'M mutation was placed in β4 and co-expressed with α2, α3, or α4. Functional receptors were not observed when β1L15'M subunits were co-expressed with other muscle nAChR subunits. The unique structure-activity relationship of PAMs and the α4β2L15'M receptor compared to α7, and the availability of high resolution α4β2 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 positive allosteric modulators (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 alpha subunits reduces heteromeric receptor channel activation, while the same mutation in neuronal β subunits allows heteromeric receptors to respond to select α7 PAMs. The results indicate a key role for this residue in the functional differences in the two main classes of neuronal nAChR.


acetylcholine receptors; allosterism; ligand gated ion channels; nicotinic acetylcholine receptors; receptor desensitization; receptor mutagenesis; receptor structure; xenopus oocytes

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