?-Conotoxins Identify the ?3?4* Subtype as the Predominant Nicotinic Acetylcholine Receptor Expressed in Human Adrenal Chromaffin Cells.

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

SUBMITTER: Hone AJ 

PROVIDER: S-EPMC4613940 | biostudies-literature | 2015 Nov

REPOSITORIES: biostudies-literature