Expression and function of two nicotinic subunits in insect neurons

Abstract

Nicotinic acetylcholine receptors (nAChRs) in insects are neuron-specific oligomeric proteins essential for the central transmission of sensory information. Little is known about their subunit composition because it is difficult to express functional insect nAChRs in heterologous systems. As an alternative approach we have examined the native expression of two subunits in neurons of the nicotinic-resistant, tobacco-feeding insect Manduca sexta. Both the alpha-subunit MARA1 and the beta-subunit MARB can be detected by in situ hybridization in the majority of cultured neurons with an overlapping, but not identical, distribution. Changes in intracellular Ca(2+) evoked by nicotinic stimulation are more strongly correlated to the expression of MARA1 than MARB and are independent of cell size. Unlike the previously reported critical role of MARA1 in mediating nicotinic Ca(2+) responses, down-regulation of MARB by RNA interference (RNAi) did not reduce the number of responding neurons or the size of evoked responses, suggesting that additional subunits remain to be identified in Manduca.

Targeted disruption of genes in the Bombyx mori sex pheromone biosynthetic pathway

“…Although RNAi has been well documented in dipterans, reports detailing application of this methodology to lepidopterans are sparse (15)(16)(17)(18)(19)(20). Here we provide evidence for the potential of RNAi to dissect the molecular interactions that constitute biosynthetic pathways and demonstrate the in vivo biological relevance of the pgACBP, mgACBP, Bmpgdesat1, pgFAR, and PBANR gene products as they relate to pheromonogenesis in B. mori.…”

A steroid hormone affects sodium channel expression in Manduca central neurons

“…In lobsters and in Drosophila, non-genomic ecdysteroid actions reduce neuromuscular efficacy prior to molting (Cooper and Ruffner 1998;Ruffner et al 1999). We cannot test for steroid effects at the protein level, because no antibodies for Manduca nAChRs are available, and nAChR antibodies from several other species do not bind specifically to Manduca nAChRs (Vermehren and Trimmer 2005; own unpublished results). However, no non-genomic effects of 20E have so far been reported in Manduca, and the time course of ecdysteroid action on neuronal growth (Prugh et al 1992;Matheson and Levine 1999) and the amplitude of calcium membrane current (Grunewald and Levine 1998) is in agreement with genomic 20E effects.…”

“…As nothing is known concerning the possible control of 20E on neurotransmitter receptor expression, we have chosen the most abundant central excitatory receptor in the insect CNS, AChR. The alpha subunit MARA 1 is critical for mediating nicotinic calcium responses (Vermehren and Trimmer 2005). ISH was conducted with single-stranded RNA (ssRNA) of 0.5 kb (see above).…”

“…Recent data from cultured Manduca neurons have indicated a strongly overlapping but not identical distribution of the α-subunit MARA1 and the β-subunit MARB. Only MARA1, but not MARB, is critical for mediating nicotinic calcium responses (Vermehren et al 2001;Vermehren and Trimmer 2005), suggesting that MARA1 either forms homomeric receptors or heteromeric recptors with an as yet unidentified subunit. In Drosophila, 10 genes encode nAChR subunits, seven of which are α-subunits, and three are non α-subunits.…”

“…In situ hybridization (ISH) has shown that the amount of acetylcholine receptor alpha subunit (AChRα, MARA1; Eastham et al 1998;Vermehren and Trimmer 2005) is not affected by 20-hydroxyecdysone (20E), whereas immunocytochemistry demonstrates that the distribution of voltage-gated sodium channels (VGSCs) in differentiating cultured thoracic neurons is affected by 20E.…”