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Nicotinic acetylcholine receptors (nAChRs) play an important role as excitatory neurotransmitters in vertebrate and invertebrate species. In insects, nAChRs are the site of action of commercially important insecticides and, as a consequence, there is considerable interest in examining their functional properties. However, problems have been encountered in the successful functional expression of insect nAChRs, although a number of strategies have been developed in an attempt to overcome such difficulties. Ten nAChR subunits have been identified in the model insect Drosophila melanogaster (Dα1-Dα7 and Dβ1-Dβ3) and a similar number have been identified in other insect species. The focus of the present study is the Dα5, Dα6 and Dα7 subunits, which are distinguished by their sequence similarity to one another and also by their close similarity to the vertebrate α7 nAChR subunit. A full-length cDNA clone encoding the Drosophila nAChR Dα5 subunit has been isolated and the properties of Dα5-, Dα6- and Dα7-containing nAChRs examined in a variety of cell expression systems. We have demonstrated the functional expression, as homomeric nAChRs, of the Dα5 and Dα7 subunits in Xenopus oocytes by their co-expression with the molecular chaperone RIC-3. Also, using a similar approach, we have demonstrated the functional expression of a heteromeric 'triplet' nAChR (Dα5 + Dα6 + Dα7) with substantially higher apparent affinity for acetylcholine than is seen with other subunit combinations. In addition, specific cell-surface binding of [125I]-α-bungarotoxin was detected in both Drosophila and mammalian cell lines when Dα5 was co-expressed with Dα6 and RIC-3. In contrast, co-expression of additional subunits (including Dα7) with Dα5 and Dα6 prevented specific binding of [125I]-α-bungarotoxin in cell lines, suggesting that co-assembly with other nAChR subunits can block maturation of correctly folded nAChRs in some cellular environments. Data are presented demonstrating the ability of the Drosophila Dα5 and Dα7 subunits to generate functional homomeric and also heteromeric nAChRs.
Figure 1. Radioligand binding to nAChR subunit chimeras expressed inâDrosophilaâS2 cells. Cell surface [125I]-α-bungarotoxin binding to transiently transfected Drosophila S2 cells with subunit chimeras (Dα5/5HT3A, Dα6/5HT3A and Dα7/5HT3A). Experiments were performed in triplicate and are meansâ±âSEM of 4â14 independent experiments.
Figure 2. Radioligand binding toâDrosophilaânAChR subunit combinations in cultured cell lines. Cell surface [125I]-α-bungarotoxin binding to cell lines transiently transfected with combinations of Dα5, Dα6 and Dα7 subunits. In all cases, subunit combinations were co-transfected with either CeRIC-3 (filled bars) or DmRIC-3 (open bars). No specific binding was detected for any subunit combination in the absence of RIC-3 (not shown). Data are presented for Drosophila S2 cells (A) and for human tsA201 cells cultured at 25â°C (B). Controls represent mock-transfected cells. Experiments were performed in triplicate and are meansâ±âSEM of 5â8 independent experiments.
Figure 3. Functional expression ofâDrosophilaânAChR subunit combinations inâXenopusâoocytes. A) Doseâresponse curves for acetylcholine are shown for homomeric Dα5 nAChRs (open circles) homomeric Dα7 nAChRs (open squares) and for triplet Dα5â+âDα6â+âDα7 nAChRs (closed circles). B) Doseâresponse curves for acetylcholine are shown for heteromeric Dα5â+âDα6 nAChRs (open circles) and Dα5â+âDα7 nAChRs (closed circles) In all cases, nAChR subunits were co-expressed with CeRIC-3. Data are meansâ±âSEM of 3â8 independent experiments.
Figure 4. Antagonism of Dα5 nAChRs by α-bungarotoxin. A) Representative responses to acetylcholine (100âμM; black bar) are shown (left), together with block after a 10âmin pre-incubation with α-bungarotoxin (100 nM; grey bar) (middle). Recovery from α-bungarotoxin block after 10 minutes is also illustrated (right). Data shown are for homomeric Dα5 nAChRs (co-expressed with CeRIC-3) but the results (complete block with full recovery) were observed for all subunit combinations that generated functional nAChRs (see Table 2). B) Data indicating the time course for recovery after a 10âmin incubation with α-bungarotoxin (100 nM; illustrated by the grey bar). Data points are normalised to the maximum response prior to block by α-bungarotoxin and are meansâ±âSEM of 3 independent experiments.
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