Hone Arik J, McIntosh J Michael, Azam Layla, Lindstrom Jon, Lucero Linda, Whiteaker Paul, Passas Juan, Blázquez Jesús, Albillos Almudena
Departamento de Farmacología y Terapéutica, Universidad Autónoma de Madrid, Madrid, Spain (A.J.H., A.A.); Departments of Biology and Psychiatry, University of Utah, Salt Lake City, Utah (J.M.M., L.A.); George E. Whalen Veterans Affairs Medical Center, Salt Lake City, Utah (J.M.M.); Department of Neuroscience, University of Pennsylvania Medical School, Philadelphia, Pennsylvania (J.L.); Division of Neurobiology, Barrow Neurological Institute, Phoenix, Arizona (L.L., P.W.); Hospital Doce de Octubre, Madrid, Spain (J.P.); and Hospital Clínico San Carlos Madrid, Spain (J.B.).
Departamento de Farmacología y Terapéutica, Universidad Autónoma de Madrid, Madrid, Spain (A.J.H., A.A.); Departments of Biology and Psychiatry, University of Utah, Salt Lake City, Utah (J.M.M., L.A.); George E. Whalen Veterans Affairs Medical Center, Salt Lake City, Utah (J.M.M.); Department of Neuroscience, University of Pennsylvania Medical School, Philadelphia, Pennsylvania (J.L.); Division of Neurobiology, Barrow Neurological Institute, Phoenix, Arizona (L.L., P.W.); Hospital Doce de Octubre, Madrid, Spain (J.P.); and Hospital Clínico San Carlos Madrid, Spain (J.B.)
Mol Pharmacol. 2015 Nov;88(5):881-93. doi: 10.1124/mol.115.100982. Epub 2015 Sep 1.
Ligands that selectively inhibit human α3β2 and α6β2 nicotinic acetylcholine receptor (nAChRs) and not the closely related α3β4 and α6β4 subtypes are lacking. Current α-conotoxins (α-Ctxs) that discriminate among these nAChR subtypes in rat fail to discriminate among the human receptor homologs. In this study, we describe the development of α-Ctx LvIA(N9R,V10A) that is 3000-fold more potent on oocyte-expressed human α3β2 than α3β4 and 165-fold more potent on human α6/α3β2β3 than α6/α3β4 nAChRs. This analog was used in conjuction with three other α-Ctx analogs and patch-clamp electrophysiology to characterize the nAChR subtypes expressed by human adrenal chromaffin cells. LvIA(N9R,V10A) showed little effect on the acetylcholine-evoked currents in these cells at concentrations expected to inhibit nAChRs with β2 ligand-binding sites. In contrast, the β4-selective α-Ctx BuIA(T5A,P6O) inhibited >98% of the acetylcholine-evoked current, indicating that most of the heteromeric receptors contained β4 ligand-binding sites. Additional studies using the α6-selective α-Ctx PeIA(A7V,S9H,V10A,N11R,E14A) indicated that the predominant heteromeric nAChR expressed by human adrenal chromaffin cells is the α3β4* subtype (asterisk indicates the possible presence of additional subunits). This conclusion was supported by polymerase chain reaction experiments of human adrenal medulla gland and of cultured human adrenal chromaffin cells that demonstrated prominent expression of RNAs for α3, α5, α7, β2, and β4 subunits and a low abundance of RNAs for α2, α4, α6, and α10 subunits.
目前缺乏能选择性抑制人类α3β2和α6β2烟碱型乙酰胆碱受体（nAChRs）而不抑制密切相关的α3β4和α6β4亚型的配体。当前在大鼠中能区分这些nAChR亚型的α-芋螺毒素（α-Ctxs）无法区分人类受体同源物。在本研究中，我们描述了α-Ctx LvIA(N9R,V10A)的研发情况，其对卵母细胞表达的人类α3β2的效力比对α3β4高3000倍，对人类α6/α3β2β3的效力比对α6/α3β4 nAChRs高165倍。该类似物与其他三种α-Ctx类似物及膜片钳电生理学方法结合使用，以表征人类肾上腺嗜铬细胞表达的nAChR亚型。在预期能抑制具有β2配体结合位点的nAChRs的浓度下，LvIA(N9R,V10A)对这些细胞中乙酰胆碱诱发的电流几乎没有影响。相反，β4选择性的α-Ctx BuIA(T5A,P6O)抑制了>98%的乙酰胆碱诱发电流，表明大多数异聚体受体含有β4配体结合位点。使用α6选择性的α-Ctx PeIA(A7V,S9H,V10A,N11R,E14A)的进一步研究表明，人类肾上腺嗜铬细胞表达的主要异聚体nAChR是α3β4*亚型（星号表示可能存在其他亚基）。人类肾上腺髓质及培养的人类肾上腺嗜铬细胞的聚合酶链反应实验支持了这一结论，该实验表明α3、α5、α7、β2和β4亚基的RNA表达显著，而α2、α4、α6和α10亚基的RNA丰度较低。
