title: Photolabeling a Nicotinic Acetylcholine Receptor (nAChR) with an (α4)3(β2)2 nAChR-Selective Positive Allosteric Modulator.
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 theα4 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 [(3)H]CMPI upon photolysis at 312 nm to identify its binding sites inTorpedonAChRs. Recording fromXenopusoocytes, we found that CMPI potentiated maximally the responses of (α4)3(β2)2nAChR to 10μM ACh (EC10) by 400% and with anEC50of ∼1µM. CMPI produced a left shift of the ACh concentration-response curve without altering ACh efficacy. In contrast, CMPI inhibited (∼35% at 10µM) ACh responses of (α4)2(β2)3nAChRs and fully inhibited human muscle andTorpedonAChRs with IC50values of ∼0.5µM. Upon irradiation at 312 nm, [(3)H]CMPI photoincorporated into eachTorpedo[(α1)2β1γδ] nAChR subunit. Sequencing of peptide fragments isolated from [(3)H]CMPI-photolabeled nAChR subunits established photolabeling of amino acids contributing to the ACh binding sites (αTyr(190),αTyr(198),γTrp(55),γTyr(111),γTyr(117),δTrp(57)) that was fully inhibitable by agonist and lower-efficiency, state-dependent [(3)H]CMPI photolabeling within the ion channel. Our results establish that CMPI is a potent potentiator of nAChRs containing anα4:α4 subunit interface, and that its intrinsic photoreactivy makes it of potential use to identify its binding sites in the (α4)3(β2)2nAChR.
authors.affiliation: Department of Pharmaceutical Sciences, Rangel College of Pharmacy, Texas A&M Health Sciences Center, Kingsville, Texas (A.K.H., F.D., Z.-J.W.); and Department of Neurobiology, Harvard Medical School, Boston, Massachusetts (A.K.H., J.B.C.) hamouda@tamhsc.edu.
authors.forename: Ayman K
authors.initials: AK
authors.lastname: Hamouda
authors.affiliation: Department of Pharmaceutical Sciences, Rangel College of Pharmacy, Texas A&M Health Sciences Center, Kingsville, Texas (A.K.H., F.D., Z.-J.W.); and Department of Neurobiology, Harvard Medical School, Boston, Massachusetts (A.K.H., J.B.C.).
authors.forename: Farah
authors.initials: F
authors.lastname: Deba
authors.affiliation: Department of Pharmaceutical Sciences, Rangel College of Pharmacy, Texas A&M Health Sciences Center, Kingsville, Texas (A.K.H., F.D., Z.-J.W.); and Department of Neurobiology, Harvard Medical School, Boston, Massachusetts (A.K.H., J.B.C.).
authors.forename: Ze-Jun
authors.initials: ZJ
authors.lastname: Wang
authors.affiliation: Department of Pharmaceutical Sciences, Rangel College of Pharmacy, Texas A&M Health Sciences Center, Kingsville, Texas (A.K.H., F.D., Z.-J.W.); and Department of Neurobiology, Harvard Medical School, Boston, Massachusetts (A.K.H., J.B.C.).
authors.forename: Jonathan B
authors.initials: JB
authors.lastname: Cohen
mesh_terms: D000494:Allosteric Regulation / Q000187:drug effects; D000818:Animals; D001665:Binding Sites; D006801:Humans; D007555:Isoxazoles / Q000494:pharmacology; D007700:Kinetics; D008958:Models, Molecular; D009124:Muscle Proteins / Q000819:agonists / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D009419:Nerve Tissue Proteins / Q000819:agonists / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D018722:Nicotinic Agonists / Q000494:pharmacology*; D018733:Nicotinic Antagonists / Q000494:pharmacology*; D009865:Oocytes / Q000187:drug effects / Q000378:metabolism; D018408:Patch-Clamp Techniques; D010782:Photolysis; D011487:Protein Conformation; D020033:Protein Isoforms / Q000819:agonists / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D021122:Protein Subunits / Q000819:agonists / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D011720:Pyrazoles / Q000494:pharmacology; D011869:Radioligand Assay; D011978:Receptors, Nicotinic / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D014101:Torpedo; D014982:Xenopus laevis
chemical_list: C000609740:3-(2-chlorophenyl)-5-(5-methyl-1-(piperidin-4-yl)-1H-pyrazol-4-yl)isoxazole; D007555:Isoxazoles; D009124:Muscle Proteins; D009419:Nerve Tissue Proteins; D018722:Nicotinic Agonists; D018733:Nicotinic Antagonists; D020033:Protein Isoforms; D021122:Protein Subunits; D011720:Pyrazoles; D011978:Receptors, Nicotinic; D011994:Recombinant Proteins