Complex Intracellular Messenger Pathways Regulate One Type of Neuronal a-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 a-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 mM a-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 a-conotoxin ImI). This indicated that two types of a-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 mM), 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 mM) 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. The nicotinic acetylcholine receptor (nAChR) has served as a model system for the study of the structure, function, and regulation of ligand-gated ion channels in vertebrates as well as in invertebrates (Osborne, 1996; Mongan et al., 1998; Swope et al., 1999). In vertebrates, nAChRs can be subdivided into two subgroups, end-plate nAChRs and neuronal nAChRs. To date, 10 known members of the gene family encode the subunits of neuronal nAChR [i.e., eight a subunits (a22a9) and three b subunits] and different neuronal nAChRs can be formed from the members of this gene family (Cordero-Erausquin et al., 2000). These nAChRs can be further classified according to their sensitivity to a-bungarotoxin into a-bungarotoxin-sensitive and -insensitive nAChRs. The nAChRs formed of a7, a8, and a9 subunits are blocked by a-bungarotoxin, whereas those composed of a2 to a6 and b2 to b4 subunits are a-bungarotoxin-resistant (Cordero-Erausquin et al., 2000). The regulation of nAChR function by intracellular messengers plays a key role in the modulation of neuronal activity (e.g., Paterson and Nordberg, 2000). Abundant evidence indicates that the nAChR is a phosphoprotein that has been shown to be phosphorylated and regulated by protein kinases such as cAMP-dependent protein kinase (PKA), protein kinase C, calcium-calmodulindependent protein kinase (CaM kinase), and endogenous protein tyrosine kinase (Margiotta et al., 1987; Eilers et al., 1997; Liu and Berg, 1999; Paterson and Nordberg, 2000). In addition, protein phosphatases, such as phosphatases PP1/ R.C. is supported by a doctoral fellowship of the Ministère de l’Education Nationale. ABBREVIATIONS: nAChR, nicotinic acetylcholine receptor; ACh, acetylcholine; PKA, cAMP-dependent protein kinase; CaM kinase, calcium-calmodulin-dependent protein kinase; DUM, dorsal unpaired median; TAG, terminal abdominal ganglia; H89, N-2[-(p-bromocinnamyl-amino)ethyl-]5-isoquinolinesulfonamide; AMP-PNP, 59-adenylyl-b-g-imidophosphate; IBMX, 3-isobutyl-1-methylxanthine; W7, N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide hydrochloride; PDE, phosphodiesterase; KN-62, 1-[N,O-Bis(5-isoquinolinesulfonyl)-N-methyl-L-tyrosil]-4-phenylpiperazine. 0026-895X/01/6001-80–91$3.00 MOLECULAR PHARMACOLOGY Vol. 60, No. 1 Copyright © 2001 The American Society for Pharmacology and Experimental Therapeutics 834/908670 Mol Pharmacol 60:80–91, 2001 Printed in U.S.A. 80 at A PE T Jornals on N ovem er 7, 2017 m oharm .aspeurnals.org D ow nladed from 2A, calcineurin, and tyrosine phosphatases, may also regulate the physiological function of nAChRs (Eilers et al., 1997; Khiroug et al., 1998; Liu and Berg, 1999). In contrast to vertebrates neuronal nAChRs, phosphorylation/dephosphorylation of nAChRs in insect neuronal preparations has not been examined in detail. It is known that acetylcholine (ACh) is the predominant excitatory neurotransmitter in insect central nervous system (Sattelle, 1985). Among insect AChRs, both a-bungarotoxin-sensitive and -insensitive nAChRs have been widely studied in their pharmacological and physiological aspects (Lapied et al., 1990; Benson, 1992; David and Pitman, 1993; Grolleau et al., 1996; Osborne, 1996). Although the molecular structure of insect nAChRs is not as well characterized as that of their vertebrates counterparts, many recent studies have focused on characterizing the subunit composition of insect nAChRs. Although several different nAChR a-type and b-type subunit genes and cDNAs have been isolated from various insect species, such as the fruit fly Drosophila melanogaster (Gundelfinger and Schulz, 2000; Lansdell and Millar, 2000a), the tobacco hornworm Manduca sexta (Eastham et al., 1998), the locusts Schistocerca gregaria and Locusta migratoria (Marshall et al., 1990; Hermsen et al., 1998), and the aphid Myzus persicae (Sgard et al., 1998; Huang et al., 2000), the manner in which these native insect nAChRs can be modulated by phosphorylation and/or dephosphorylation induced by protein kinases and protein phosphatases remains unknown. Because of the importance of insect nAChRs as target sites for the major highly effective and widely used neonicotinoid insecticides, such as imidacloprid (Yamamoto and Casida, 1999), it seems necessary to better understand the intracellular messenger pathways involved in the regulation of native nAChRs. Such unknown intracellular mechanisms underlying the nAChR functional properties should undoubtedly alter the mode of action of this new class of insecticides. Consequently, in this study, we have begun to study, for the first time in insect neuronal preparations, the phosphorylation/dephosphorylation mechanism involved in the regulation of the a-bungarotoxin–insensitive neuronal nAChRs using whole-cell, patch-clamp technique. We have found that two pharmacologically distinct types of native somatic a-bungarotoxin–insensitive nAChRs are differentially modulated by complex intracellular mechanisms involving PKA, an okadaic acid-sensitive protein phosphatase, and CaM kinase II. The possible cross talk between these intracellular messenger cascades is discussed. Moreover, we also report, for the first time, that phosphorylation/dephosphorylation process could strongly affect the mode of action of imidacloprid, known to act as agonist at the cockroach DUM neuron nAChRs (Buckingham et al., 1997). Materials and Methods Preparation. Experiments were performed on DUM neuron cell bodies isolated from the midline of the terminal abdominal ganglia (TAG) of the nerve chord of adult male cockroaches (Periplaneta americana) obtained from our laboratory stock colony maintained at 29°C on 12-h light/dark cycle. Animals were immobilized ventral side up on a dissection dish. The ventral cuticle and the accessory gland were removed to allow access to the TAG. The abdominal nerve cord and its TAG, carefully dissected under a binocular microscope, were placed in normal cockroach saline containing 200 mM NaCl, 3.1 mM KCl, 5 mM CaCl2, 4 mM MgCl2, 50 mM sucrose, 10 mM HEPES; pH was adjusted to 7.4 with NaOH. Cell Isolation. Isolation of adult DUM neuron cell bodies was performed under sterile conditions using enzymatic digestion and mechanical dissociation of the median part of the TAG as described previously (Lapied et al., 1989). DUM neuron cell bodies were maintained at 29°C for 24 h before electrophysiological experiments were carried out. The DUM neuron cell bodies used in the present study were chosen as indicated previously (Lapied et al., 1989). Whole-Cell Recording and Data Analysis. Nicotine-, acetylcholineand imidacloprid-induced ionic currents were recorded using the patch-clamp technique in the whole-cell recording configuration (Hamill et al., 1981) under voltage-clamp mode. Signals were recorded with an Axopatch 200A patch-clamp amplifier (Axon Instruments, Foster City, CA). Patch pipettes were pulled from borosilicate glass capillary tubes (GC 150T-10; Clark Electromedical Instruments, Harvard Apparatus, Edenbridge, UK) using a PP83 puller (Narishige, Tokyo, Japan). Pipettes had resistances ranging from 0.8 to 1 MV when filled with internal solutions (see composition below). The liquid junction potential between bath and internal solutions was always corrected before the formation of a gigaOhm seal (.5 GV). Ionic currents induced by the cholinergic agonists were recorded on an NEC Celeron 333 computer with software control pClamp (version 6.03; Axon Instruments) connected to a 125-kHz Labmaster DMA data acquisition system (TL-1–125 interface; Axon Instruments). DUM neuron cell bodies were voltage-clamped at a steady-state holding potential of 250 mV (except when otherwise