Unraveling amino acid residues critical for allosteric potentiation of (α4)3(β2) 2-type nicotinic acetylcholine receptor responses

Neuronal nicotinic acetylcholine receptors (nAChRs) are promising drug targets to manage several neurological disorders and nicotine addiction. Growing evidence indicates that positive allosteric modulators of nAChRs improve pharmacological specificity by binding to unique sites present only in a subpopulation of nAChRs. Furthermore, nAChR positive allosteric modulators such as NS9283 and CMPI have been shown to potentiate responses of (alpha 4)3(beta 2)2 but not (alpha 4)2(beta 2)3 nAChR isoforms. This selective potentiation underlines that the alpha 4: alpha 4 interface, which is present only in the (alpha 4)3(beta 2)2 nAChR, is an important and promising drug target. In this report we used site-directed mutagenesis to substitute specific amino acid residues and computational analyses to elucidate CMPI's binding mode at the alpha 4: alpha 4 subunit extracellular interface and identified a unique set of amino acid residues that determined its affinity. We found that amino acid residues alpha 4Gly-41, alpha 4Lys-64, and alpha 4Thr-66 were critical for (alpha 4)3(beta 2)2 nAChR potentiation by CMPI, but not by NS9283, whereas amino acid substitution at alpha 4His-116, a known determinant of NS9283 and of agonist binding at the alpha 4: alpha 4 subunit interface, did not reduce CMPI potentiation. In contrast, substitutions at alpha 4Gln-124 and alpha 4Thr-126 reduced potentiation by CMPI and NS9283, indicating that their binding sites partially overlap. These results delineate the role of amino acid residues contributing to the alpha 4: alpha 4 subunit extracellular interface in nAChR potentiation. These findings also provide structural information that will facilitate the structure-based design of novel therapeutics that target selectively the (alpha 4)3(beta 2)2 nAChR.