Abstract

Positive allosteric modulators (PAMs) of nicotinic acetylcholine (ACh) 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. 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. In addition, we use the intrinsic reactivity of [³H]CMPI upon photolysis at 312 nm to identify its binding sites in Torpedo nAChRs. Recording from Xenopus oocytes, we found that CMPI potentiated maximally the responses of (α4)₃(β2)₂ nAChR to 10 μM ACh (EC₁₀) by 400% and with an EC₅₀ of ∼1 µM. 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 IC₅₀ values 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 that 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.