Splice-variant- and stage-specific RNA editing of the Drosophila GABA receptor modulates agonist potency.

The molecular diversity of many gene products functioning in the nervous system is enhanced by alternative splicing and adenosine-to-inosine editing of pre-mRNA. Using RDL, a Drosophila melanogaster GABA-gated ion channel, we examined the functional impact of RNA editing at several sites along with alternative splicing of more than one exon. We show that alternative splicing and RNA editing have a combined influence on the potency of the neurotransmitter GABA, and the editing isoforms detected in vivo span the entire functional range of potencies seen for all possible edit variants expressed in Xenopus laevis oocytes. The extent of RNA editing is developmentally regulated and can also be linked to the choice of alternative exons. These results provide insights into how the rich diversity of signaling necessary for complex brain function can be achieved by relatively few genes.

Role in the selectivity of neonicotinoids of insect-specific basic residues in loop D of the nicotinic acetylcholine receptor agonist binding site.

The insecticide imidacloprid and structurally related neonicotinoids act selectively on insect nicotinic acetylcholine receptors (nAChRs). To investigate the mechanism of neonicotinoid selectivity, we have examined the effects of mutations to basic amino acid residues in loop D of the nAChR acetylcholine (ACh) binding site on the interactions with imidacloprid. The receptors investigated are the recombinant chicken alpha4beta2 nAChR and Drosophila melanogaster Dalpha2/chicken beta2 hybrid nAChR expressed in Xenopus laevis oocytes. Although mutations of Thr77 in loop D of the beta2 subunit resulted in a barely detectable effect on the imidacloprid concentration-response curve for the alpha4beta2 nAChR, T77R;E79V double mutations shifted the curve dramatically to higher affinity binding of imidacloprid. Likewise, T77K;E79R and T77N;E79R double mutations in the Dalpha2beta2 nAChR also resulted in a shift to a higher affinity for imidacloprid, which exceeded that observed for a single mutation of Thr77 to basic residues. By contrast, these double mutations scarcely influenced the ACh concentration-response curve, suggesting selective interactions with imidacloprid of the newly introduced basic residues. Computational, homology models of the agonist binding domain of the wild-type and mutant alpha4beta2 and Dalpha2beta2 nAChRs with imidacloprid bound were generated based on the crystal structures of acetylcholine binding proteins of Lymnaea stagnalis and Aplysia californica. The models indicate that the nitro group of imidacloprid interacts directly with the introduced basic residues at position 77, whereas those at position 79 either prevent or permit such interactions depending on their electrostatic properties, thereby explaining the observed functional changes resulting from site-directed mutagenesis.

Alpha7 mutants mimicking atypical motifs (YxxCC of loop-C, and E to H at -1' in TM2) in the C. elegans LEV-8 subunit affect nicotinic acetylcholine receptor function.

The ACR-8-like group of C. elegans nicotinic acetylcholine receptor (nAChR) subunits contain unusual motifs in the ACh binding site and in the -1' position of transmembrane region two (TM2). Using site-directed mutagenesis (SDM) we have introduced these motifs into chicken alpha7 as it has not been possible to express C. elegans nAChR in vitro. Oocytes expressing alpha7 with the C. elegans binding motif show a reduced affinity and efficacy for both ACh and nicotine. The blocking action of the anthelmintic drug levamisole is reduced. The TM2 motif resulted in a non-functional receptor. We conclude that the TM2 motif profoundly restricts cation movement through the alpha7 channel but does not confer anion permeability. The altered form of the ACh binding motif is likely to result in a receptor with altered pharmacology, adding potential functional diversity at synapses in the nervous system and neuromuscular junctions of C. elegans.

Insect-vertebrate chimeric nicotinic acetylcholine receptors identify a region, loop B to the N-terminus of the Drosophila Dalpha2 subunit, which contributes to neonicotinoid sensitivity.

A chimera based on the chicken alpha4 nicotinic acetylcholine receptor (nAChR) subunit containing an insert from loop B to the N-terminus of the Drosophila melanogaster Dalpha2 (=SAD) subunit was constructed and co-expressed with the chicken beta2 nAChR subunit in Xenopus laevis oocytes. The actions of the neonicotinoid insecticide imidacloprid were examined. Replacement of the region loop B to the N-terminus of the alpha4 subunit by the corresponding region of the Dalpha2 subunit had little effect on the concentration-response curve for imidacloprid. However, replacement of Glu219 by proline in the YXCC motif in loop C of the chimeric alpha4 subunit resulted in a marked displacement to the left of the concentration-response curve for imidacloprid not seen when an equivalent mutation was made in the alpha4beta2 nAChR. The results suggest that the region loop B to the N-terminus in the Dalpha2 subunit contributes to the high imidacloprid sensitivity of the hybrid Dalpha2beta2 nAChR.

Roles of loop C and the loop B-C interval of the nicotinic receptor alpha subunit in its selective interactions with imidacloprid in insects.

To elucidate the mechanism of selective action of imidacloprid on insect nicotinic acetylcholine receptors (nAChRs), we examined the roles of loop C and the loop B-C interval region in receptor interactions with imidacloprid. The P242E mutation in loop C of the Drosophila SAD subunit (the second alpha-like Drosophila nicotinic acetylcholine receptor subunit, also called Dalpha2 subunit) reduced imidacloprid sensitivity of the SAD-chicken beta2 hybrid nAChR, whereas the E219P mutation of the alpha4 subunit increased the imidacloprid sensitivity of the alpha4beta2 nAChR. Deletion of the loop B-C interval region from the SAD subunit enhanced the effect of the P242E mutation on the SADbeta2 hybrid nAChR, suggesting important roles of the regions investigated in the nAChR-imidacloprid interactions.

Combinatorial mutations in loops D and F strongly influence responses of the alpha7 nicotinic acetylcholine receptor to imidacloprid.

The nitro group of a neonicotinoid, imidacloprid, plays a key role in its selective actions on insect nicotinic acetylcholine receptors (nicotinic AChRs) and is postulated to bind close to residues Q79 in loop D and G189 in loop F of the chicken alpha7 nicotinic AChR. To evaluate the relative contributions of these residues to interactions with imidacloprid, Q79 and G189 were replaced in tandem by first basic then acidic residues. Changes in the currents evoked by imidacloprid and acetylcholine (ACh) on the alpha7 wild type and mutant receptors expressed in Xenopus laevis oocytes were investigated using two-electrode voltage clamp electrophysiology. An increase in the efficacy of imidacloprid for the alpha7 receptor resulting from the Q79K and Q79R mutations was suppressed by a G189E mutation in loop F. However, the increases in efficacy resulting from such Q79 mutations were scarcely influenced by a G189D substitution. Three-dimensional modeling of the alpha7 nicotinic AChR, based on the acetylcholine-binding protein (AChBP) of Lymnaea stagnalis, suggests that the reduced efficacy of imidacloprid following the G189E mutation is likely to result from carboxylate interference with the electronic interactions between the nitro group of imidacloprid and the basic residues in loop D.