Molecular biology and electrophysiology of neuronal nicotinic receptors of rat chromaffin cells

Neuronal nicotinic acetylcholine receptors of chromaffin cells in the adrenal medulla are physiologically activated by acetylcholine to mediate catecholamine release into the bloodstream. The present study examined the subunit composition and functional properties of rat chromaffin cell neuronal nicotinic acetylcholine receptors using molecular biology, immunocytochemistry and whole-cell patchclamp. Reverse transcription-polymerase chain reaction analysis indicated the presence of α2, α3, α4, α5, α7, β2 and β4 transcripts (α6 and β3 could not be detected). Immunocytochemistry revealed most cells positive for α3, β2, β4 and α5 proteins. Few cells were immunoreactive for α2 and α4, while none was for α7. At single-cell level, colocalization could be demonstrated for α3α5 and α4β2. Western blot analysis confirmed antibody specificity for α3, α4, β5, β2 and β4 subunits. Inward currents elicited by nicotine pulses were insensitive to α-bungarotoxin and low doses of methyllycaconitine, demonstrating lack of functional α7 receptors. Partial block of nicotine currents was observed with either AuIB α-conotoxin (selective against α3β4 receptors) or MII α-conotoxin (selective against α3β2 receptors). With high concentrations of co-applied toxins, antagonism occlusion developed, suggesting loss of subunit selectivity. Antagonism by dihydro-β-erythroidine summated nonlinearly with AuIB and MII inhibition, confirming heterogeneity of neuronal nicotinic acetylcholine receptor block. The present results suggest that the most frequently encountered receptors of rat chromaffin cells should comprise α3β4, α3β2 with the addition of α5 subunits. Because of the prevailing subunit composition, rat chromaffin cell neuronal nicotinic acetylcholine receptors are suitable models, particularly for the α3β4 subclasses of mammalian brain receptors recently demonstrated in discrete cerebral areas.