TY - JOUR T1 - Photolabeling a Nicotinic Acetylcholine Receptor (nAChR) with an (α4)3(β2)2 nAChR-selective Positive Allosteric Modulator JF - Molecular Pharmacology JO - Mol Pharmacol DO - 10.1124/mol.116.103341 SP - mol.116.103341 AU - Ayman K Hamouda AU - Farah Deba AU - Ze-Jun Wang AU - Jonathan B. Cohen Y1 - 2016/01/01 UR - http://molpharm.aspetjournals.org/content/early/2016/03/14/mol.116.103341.abstract N2 - Positive allosteric modulators (PAMs) of nicotinic acetylcholine receptors (nAChRs) have potential clinical applications in the treatment of nicotine dependence and many neuropsychiatric conditions associated with decreased brain cholinergic activity, and 3-(2-chlorophenyl)-5-(5-methyl-1-(piperidin-4-yl)-1H-pyrrazol-4-yl)isoxazole (CMPI) has been identified as a PAM selective for neuronal nAChRs containing the α4 subunit (Albrecht et al., Biorg. Med. Chem. Lett. 18: 5209-5212 (2008)). In this report, we compare CMPI interactions with low-sensitivity (α4)3(β2)2 and high-sensitivity (α4)2(β2)3 nAChRs and with muscle-type nAChRs, and we use the intrinsic photoreactivity of [3H]CMPI to identify its binding sites in Torpedo nAChRs. Recording from Xenopus oocytes, we found that CMPI potentiated maximally by 400% and with an EC50 of ~1 μM the responses of (α4)3(β2)2 nAChRs to ACh at 10 μM (EC10). CMPI produced a left-shift of the ACh concentration-response curve without altering ACh efficacy. In contrast, CMPI inhibited (~35% at 10 μM) ACh responses of (α4)2(β2)3 nAChRs and fully inhibited human muscle and Torpedo nAChRs with IC50s of ~0.5 μM. Upon irradiation at 312 nm, [3H]CMPI photoincorporated into each Torpedo ((α1)2β1γδ) nAChR subunit. Sequencing of peptide fragments isolated from [3H]CMPI-photolabeled nAChR subunits established photolabeling of amino acids contributing to the ACh binding sites (αTyr190, αTyr198, γTrp55, γTyr111, γTyr117, δTrp57) that was fully inhibitable by agonist and lower efficiency, state-dependent [3H]CMPI photolabeling within the ion channel. Our results establish CMPI is a potent potentiator of nAChRs containing an α4:α4 subunit interface and that its intrinsic photoreactivy makes it of potential use to identify its binding sites in the (α4)3(β2)2 nAChR. ER -