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Cellular responses to Ca(2+) require intermediary proteins such as calcium/calmodulin-dependent protein kinase II (CaMKII), which transduces the signal into downstream effects. We recently demonstrated that the cockroach genome encodes five different CaMKII isoforms, and only PaCaMKII-E isoform is specifically expressed in the dorsal unpaired median neurosecretory cells. In the present study, using antisense oligonucleotides, we demonstrated that PaCaMKII-E isoform inhibition reduced nicotine-induced currents through α-bungarotoxin-sensitive and -insensitive nicotinic acetylcholine receptor subtypes. Specifically, PaCaMKII-E isoform is sufficient to repress nicotinic current amplitudes as a result of its depression by antisense oligonucleotides. Similar results were found using the neonicotinoid insecticide clothianidin, which acted as a full agonist of dorsal unpaired median neuron nicotinic acetylcholine receptors. Clothianidin current amplitudes are strongly reduced under bath application of PaCaMKII-E antisense oligonucleotides but no significant results are found with α-bungarotoxin co-applied, demonstrating that CaMKII-E isoform affects nicotine currents through α-bungarotoxin-sensitive and -insensitive receptor subtypes whereas clothianidin currents are reduced via α-bungarotoxin-insensitive receptors. In addition, we found that intracellular calcium increase induced by nicotine and clothianidin were reduced by PaCaMKII-E antisense oligonucleotides, demonstrating that intracellular calcium increase induced by nicotine and clothianidin are affected by PaCaMKII-E inhibition. Cellular responses to Ca(2+) require intermediary proteins such as calcium/calmodulin-dependent protein kinase II (CaMKII). We recently demonstrated that the cockroach genome encodes five different CaMKII isoforms and only PaCaMKII-E isoform was specifically expressed in the dorsal unpaired median neurosecretory cells. Here we show that specific inhibition of PaCaMKII-E isoform is associated with a decrease in nicotine- and clothianidin-induced currents. In addition, analysis of calcium changes demonstrates that PaCaMKII-E inhibition induces a decrease in intracellular calcium concentration., (© 2014 International Society for Neurochemistry.)

The goal of the present study is to examine the agonist action of nornicotine on insect nicotinic acetylcholine receptors. Using patch-clamp techniques on cockroach dorsal unpaired median neurons, we demonstrated that nornicotine induced two distinct ionic currents named types 1 and 2. We found that alpha-bungarotoxin induced a rapid desensitization of type 1 currents whereas type 2 was completely blocked. Interestingly, types 1 and 2 currents were not blocked by the muscarinic antagonist, pirenzepine but by co-application of 1 μM pirenzepine and 0.5 μM alpha-bungarotoxin, suggesting that muscarinic receptors modulated nornicotine-induced current amplitudes. In addition, type 1 current amplitudes were strongly reduced by 20 μM d-tubocurarine and 5 μM mecamylamine which blocked the previously identified alpha-bungarotoxin-insensitive nAChR1 and nAChR2 receptors. Co-application of alpha-bungarotoxin with d-tubocurarine or mecamylamine completely blocked all ionic currents. We propose that types 1 and 2 currents are associated to several nicotinic receptors subtypes, including nAChR1 and nAChR2 receptors. Finally, we conclude that nornicotine could be used as an agonist to identify distinct insect nicotinic receptors., (Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.)