In the present study, we have electrophysiologically characterized native nicotinic acetylcholine receptors (nAChRs) in human chromaffin cells of the adrenal gland as well as their contribution to the exocytotic process. ?-Conotoxin AuIB blocked by 14 ± 1% the acetylcholine (ACh)-induced nicotinic current. ?-Conotoxin MII (?-Ctx MII) exhibited an almost full blockade of the nicotinic current at nanomolar concentrations (IC(50)=21.6 nM). The ?6*-preferring ?-Ctx MII mutant analogs, ?-Ctx MII[H9A,L15A] and ?-Ctx MII[S4A,E11A,L15A], blocked nAChR currents with an IC(50) of 217.8 and 33 nM, respectively. These data reveal that nAChRs in these cells include the ?6* subtype. The washout of the blockade exerted by ?-conotoxin BuIA (?-Ctx BuIA; 1 ?M) on ACh-evoked currents was slight and slow, arguing in favor of the presence of a ?4 subunit in the nAChR composition. Exocytosis was almost fully blocked by 1 ?M ?-Ctx MII, its mutant analogs, or ?-Ctx BuIA. Finally, the fluorescent analog Alexa Fluor 546-BuIA showed distinct staining in these cells. Our results reveal that ?6?4* nAChRs are expressed and contribute to exocytosis in human chromaffin cells of the adrenal gland, the main source of adrenaline under stressful situations.
