Characterization of a nicotinic acetylcholine receptor from the insect Manduca sexta

Abstract

Manduca sexta is a nicotine-insensitive insect, the larval form of which feeds on tobacco. It has been postulated that its nicotine insensitivity may reflect the presence of a modified nicotinic acetylcholine receptor whose alpha subunits lack the amino acid residues necessary for binding nicotine: we have performed ligand binding assays and molecular cloning to examine this hypothesis. [125I]alpha-bungarotoxin bound specifically to both larval and adult membranes, with Kd values of 7.6 and 6.5 nM and Bmax values of 119 and 815 fmol/mg protein, respectively. The pharmacological profile of [1251]alpha-bungarotoxin binding was similar in both tissues. In particular, nicotine (Ki values: 1.6 microM and 2 microM for larvae and adults, respectively) competed with an affinity similar to that found for nicotine-sensitive insects. No alpha-bungarotoxin-insensitive binding sites labelled by [3H]epibatidine could be detected. Using the alpha-like subunit from the locust Schistocerca gregaria to probe two cDNA libraries, and by inverse PCR on circularized genomic DNA from Manduca sexta, we have obtained overlapping cDNA clones that contain the complete coding sequence of a putative nicotinic subunit from Manduca sexta (MARA1). No other alpha-subunit cDNAs were isolated using this probe, although it hybridized to multiple bands on Southern blots. The sequence of MARA1 is consistent with an alpha-like subunit capable of binding alpha-bungarotoxin, and it retains all those amino acids implicated in nicotine binding to vertebrate nicotinic receptors. Taken together, these findings provide no support for the hypothesis that the nicotine insensitivity of Manduca sexta is the result of a nicotinic receptor with diminished nicotine binding.

Expression and function of two nicotinic subunits in insect neurons

“…The 5Ј untranslated region and part of the 5Ј region of the MARA1 subunit (including two introns) were initially cloned by Eastham et al (1998) using genomic DNA (gDNA). The rest of the sequence (translated region including the 3Ј untranslated region) was obtained from pupal brain complementary cDNA.…”

“…In addition, because of the importance of these receptors as targets for insecticides, extensive research has been carried out comparing the structural basis of ligand binding in insect and vertebrate receptors (Debnath et al, 2003;Tomizawa and Casida, 2003;Shimomura et al, 2003;Zhang et al, 2002). Electrophysiological and radioligand binding studies on insect neuronal nAChRs suggest a diversity of nAChRs (Eastham et al, 1998;Lind et al, 1988Lind et al, , 2001, which is supported by the cloning and sequencing of multiple genes coding for insect nicotinic subunits (Schafer, 2002). However, the subunit composition and stoichiometry of insect nAChRs are poorly understood (Tomizawa and Casida, 2001), partly because functional expression in Xenopus oocytes has been difficult Lansdell et al, 1997;Schulz et al, 2000).…”

“…The present study examined the expression and putative roles of two nAChR subunits, MARA1 (Manduca acetylcholine receptor alpha-like subunit 1) and MARB (Manduca acetylcholine receptor betalike) (Eastham et al, 1998), using in situ hybridization (ISH), ratiometric Ca 2ϩ imaging, and RNAi in neurons of Manduca sexta. The abdominal ganglia contain easily identified sensory to motoneuron synapses, which makes larval Manduca a useful model system for investigating the mechanisms of cholinergic signaling (Qazi et al, 1996;Weeks, 1991, 1993).…”

Neuronal Nicotinic Receptors in the Locust Locusta migratoria

“…A large body of evidence suggests the existence of both muscarinic and nicotinic acetylcholine (nAChR) 1 receptors in the insect brain, with nAChR-coding RNAs having been identified in several species, including the fruit fly Drosophila (2), the locust Schistocerca (3), the tobacco hornworm Manduca (4), and the peach-tomato aphid Myzus (5). Considerable pharmacological diversity of nicotinic receptors is indicated by the existence of ␣BTX-sensitive and -insensitive receptors (6,7) and by the rather wide variation of responses to nicotinic and non-nicotinic drugs of insect neurons and membrane preparations (8,9).…”

Lethal effects of an insecticidal spider venom peptide involve positive allosteric modulation of insect nicotinic acetylcholine receptors

“…A total of 10 genes have been identified in insects, nominally α1-α7 and β1-β3 in order of discovery, however in reality fewer likely form functional receptors (Jonas et al, 1990.;Lansdell and Millar, 2000;Littleton and Ganetzky, 2000;Schulz et al, 1998). Unfortunately, while the successful cloning of various nAChR subunits has been reported in several insect species, few have been functionally expressed (Eastham et al, 1998;Gao et al, 2007;Hermsen et al, 1998;Millar and Lansdell, 2010). Importantly, these insect subunits possess relatively low homology with vertebrates, most exhibiting only 30-40% homology with mammalian α2 or α7 subunits (Jones and Sattelle, 2010).…”