alpha-Conotoxins Identify the alpha 3 beta 4(star) Subtype as the Predominant Nicotinic Acetylcholine Receptor Expressed in Human Adrenal Chromaffin Cells

作者：Hone Arik J; McIntosh J Michael; Azam Layla; Lindstrom Jon; Lucero Linda; Whiteaker Paul; Passas Juan; Blazquez Jesus; Albillos Almudena^*

来源：Molecular Pharmacology, 2015, 88(5): 881-893.

DOI：10.1124/mol.115.100982

Ligands that selectively inhibit human alpha 3 beta 2 and alpha 6 beta 2 nicotinic acetylcholine receptor (nAChRs) and not the closely related alpha 3 beta 4 and alpha 6 beta 4 subtypes are lacking. Current alpha-conotoxins (alpha-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 alpha-Ctx LvIA(N9R, V10A) that is 3000-fold more potent on oocyte-expressed human alpha 3 beta 2 than alpha 3 beta 4 and 165-fold more potent on human alpha 6/alpha 3 beta 2 beta 3 than alpha 6/alpha 3 beta 4 nAChRs. This analog was used in conjuction with three other alpha-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 beta 2 ligand-binding sites. In contrast, the beta 4-selective alpha-Ctx BuIA(T5A, P6O) inhibited >98% of the acetylcholine-evoked current, indicating that most of the heteromeric receptors contained beta 4 ligand-binding sites. Additional studies using the alpha 6-selective alpha-Ctx PeIA(A7V, S9H, V10A, N11R, E14A) indicated that the predominant heteromeric nAChR expressed by human adrenal chromaffin cells is the alpha 3 beta 4* 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 alpha 3, alpha 5, alpha 7, beta 2, and beta 4 subunits and a low abundance of RNAs for alpha 2, alpha 4, alpha 6, and alpha 10 subunits.

• 出版日期2015-11