Complex intracellular messenger pathways regulate one type of neuronal alpha-bungarotoxin-resistant nicotinic acetylcholine receptors expressed in insect neurosecretory cells (dorsal unpaired median neurons).

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.