Although molecular biology provides new insights into the subunit compositions and the stoichiometries of insect neuronal
nicotinic acetylcholine receptors (nAChRs), our knowledge about the phosphorylation/dephosphorylation mechanisms of native neuronal nAChRs is limited. The regulation of
alpha-bungarotoxin-resistant nAChRs was studied on dissociated adult dorsal unpaired median neurons isolated from the terminal abdominal
ganglion of the cockroach Periplaneta americana, using whole-cell, patch-clamp technique. Under 0.5 microM
alpha-bungarotoxin treatment, pressure ejection application of
nicotine or
acetylcholine onto the cell body induced an inward current exhibiting a biphasic current-voltage relationship. We found that two distinct components underlying the biphasic curve differed in their ionic permeability and pharmacology (one being sensitive to
d-tubocurarine, and the other affected only by
mecamylamine and
alpha-conotoxin ImI). This indicated that two types of
alpha-bungarotoxin-resistant nAChRs (named nAChR1 and nAChR2) mediated the nicotinic response. These two components were also differentially sensitive to rundown and intracellular messengers. Intracellular application of 0.1 mM cAMP only increased the current amplitude mediated by nAChR1. Using
forskolin (1 microM),
W7 and
H89, we demonstrated that
adenylyl cyclase, sensitive to
calcium/
calmodulin complex, regulated nAChR1 via a cAMP/
cAMP-dependent protein kinase cascade. By contrast, internal cAMP concentration higher than 0.1 mM reduced the current amplitude. This effect, mimicked by high external concentration of
forskolin (100 microM) and
IBMX, was reversed by
okadaic acid, suggesting the implication of a
protein phosphatase. Using
KN-62, we demonstrated that
calmodulin-Kinase II also modulated directly and indirectly nAChR1, via an inhibition of the
phosphatase activity. Finally, we reported that phosphorylation/dephosphorylation of nAChR1 strongly affected the action of the widely used
neonicotinoid insecticide imidacloprid.