Nicotinic acetylcholine receptors of Drosophila: three subunits encoded by genomically linked genes can co‐assemble 
into the same receptor complex

Abstract

The second b-like subunit (SBD) is a putative structural subunit of Drosophila melanogaster nicotinic acetylcholine receptors (nAChRs). Here we have produced speci®c antibodies against SBD to study, which other nAChR subunits can co-assemble with SBD in receptor complexes of the Drosophila nervous system. Immunohistochemical studies in the adult optic lobe revealed that SBD has a distribution similar to that of the a-subunit ALS in the synaptic neuropil. The subunits ALS, Da2 and SBD can be co-puri®ed by a-bungarotoxin af®nity chromatography. Moreover, anti-SBD antibodies co-precipitate ALS and Da2 and, vice versa, ALS and Da2 antibodies co-immunoprecipitate SBD protein. Two-step immunoaf®nity chromatography with immobilized antibodies against ALS and Da2 revealed the existence of nAChR complexes that include ALS, Da2 and SBD as integral components. Interestingly, the genes encoding these three subunits appear to be directly linked in the Drosophila genome at region 96 A of the third chromosome. In addition, SBD appears to be a component of a different receptor complex, which includes the ARD protein as an additional b-subunit, but neither ALS nor Da2 nor the third a-subunit Da3. These ®ndings suggest a considerable complexity of the Drosophila nicotinic receptor system. Keywords: Drosophila genome, gene cluster, immunoprecipitation, insect nervous system, neurotransmitter receptor, quaternary protein structure. Nicotinic acetylcholine receptors (nAChRs) are ligand-gated ion channels assembled from multiple subunits. Prototypic nicotinic receptors are found at the vertebrate neuromuscular junction where they mediate excitatory neurotransmission. These muscle receptors are pentamers of known subunit composition, i.e. a 2 bcd or a 2 bed (reviewed by Galzi et al. 1991;Hucho et al. 1996). A variety of nAChR subunits (a2±a9 and b2±b4) are expressed in the vertebrate nervous system, where they assemble into multiple subtypes of heteromeric and homomeric neuronal receptors (for review see Lindstrom 2000).In the insect CNS nAChRs are widely expressed and mediate fast excitatory synaptic transmission (Sattelle 1980). Most of these receptors are thought to constitute heteromeric complexes (Gundelfinger 1992); however, the subunit composition of native nAChRs in insects is still unknown. From the fruit fly Drosophila melanogaster, six neuronal nAChR subunits have been cloned to date; these are the a-type subunits ALS, Da2/SAD, Da3, Da4 and the b-type subunits ARD and SBD (Gundelfinger and Hess 1992;Gundelfinger and Schulz 2000;Lansdell and Millar 2000). The existence of additional nAChR subunits can be predicted based on the analysis of the Drosophila genome These authors contributed equally to this work. Abbreviations used: aBgt, a-bungarotoxin; ALS, a-like subunit; ARD, acetylcholine receptor protein of Drosophila; Da2, Drosophila a-subunit 2; Da3, Drosophila a-subunit 3; Da4, Drosophila a-subunit 4; mAb, monoclonal antibody; nAChR, nicotinic acetylcholine receptor; pAb, polyclonal antibody; SBD, second b-like ...

The nicotinic acetylcholine receptor gene family of the honey bee, Apis mellifera

“…In the Apis genome, only Amel␣1 and Amel␣2 are clustered, being situated within 120 kb of each other. Immunohistochemical and coimmunoprecipitation studies show that D␣1, D␣2, and D␤2 are integral components of certain nAChR subtypes, leading to the suggestion that clustering may facilitate coordinate expression and coassembly of the nAChR subunits (Chamaon et al 2002). The separation of the Apis D␤2 ortholog Amel␣8 from the cluster may thus result in diversification of receptor expression and coassembly.…”

The Drosophila Acetylcholine Receptor Subunit Dα5 Is Part of an α-Bungarotoxin Binding Acetylcholine Receptor

“…47). Chamaon et al (17,48) probed the structure of the Drosophila nicotinic acetylcholine receptor with antibodies raised against the ALS, D␣2, D␣3, ARD, and SBD subunits. Based on their analysis, they propose the occurrence of three distinct receptor subtypes composed of either ALS, SBD, D␣2 or D␣3, ARD, or ARD, SBD with the remaining subunits for each pentameric complex to be determined.…”

“…Little information is currently available about the nature of the receptor subtypes assembled from the various subunit genes in Drosophila. Immunoprecipitation experiments indicate that the ALS and SAD subunits might be part of a receptor complex while D␣3 and ARD might be part of a different receptor subtype (17). It should be noted, however, that it has not been possible to reconstitute functional nAChRs from Drosophila in a heterologous system with any of these subunit combinations tested so far (18,19).…”

Diverse Actions and Target-Site Selectivity of Neonicotinoids: Structural Insights

“…7). This subunit combination was adopted for the following reasons: 1) both ␣2 and ␤1 subunits are important subunits and have a similar distribution in the D. melanogaster central nervous system (Jonas et al, 1994); 2) the D. melanogaster D␣2 and D␤1 subunits were copurified with the D␣1 and D␣3 subunits from the fly heads by ␣-bungarotoxin-affinity column (Chamaon et al, 2002); 3) the Mp␣2/rat ␤2 hybrid nAChR is much more sensitive to imidacloprid than the Mp␣1/rat ␤2 hybrid nAChR (Huang et al, 1999), and the Mp␣2 subunit coassembles with the Mp␤1 subunit in the D. melanogaster S2 cell (Huang et al, 2000); and 4) the Mp␣2 subunit has a valine at position X in the YXCC motif (Fig. 3), which obviates the need to consider possible hydrogen bonding with NO 2 .…”