Sequence and functional expression of a single alpha subunit of an insect nicotinic acetylcholine receptor.

Abstract

We report the isolation and sequence of a cDNA clone that encodes a locust (Schistocerca gregaria) nervous system nicotinic acetylcholine receptor (AChR) subunit (aLl). The calculated molecular weight of the unglycosylated polypeptide, which contains in the proposed extracellular domain two adjacent cysteine residues which are characteristic of a (ligand binding) subunits, is 60 641 daltons. Injection into Xenopus oocytes, of RNA synthesized from this clone in vitro, results in expression of functional nicotinic receptors in the oocyte membrane. In these, nicotine opens a cation channel; the receptors are blocked by both a-bungarotoxin (a-Bgt) and x-bungarotoxin (x-Bgt). Reversible block of the expressed insect AChR by mecamylamine, d-tubocurarine, tetraethylammonium, bicuculline and strychnine has also been observed. These data are entirely consistent with previously reported electrophysiological studies on in vivo insect nicotinic receptors and also with biochemical studies on an cx-Bgt affinity purified locust AChR. Thus, a functional receptor exhibiting the characteristic pharmacology of an in vivo insect nicotiniic AChR can be expressed in Xenopus oocytes by injection with a single subunit RNA.

Cloning and Functional Characterisation of Two Novel Nicotinic Acetylcholine Receptor α Subunits from the Insect Pest Myzus persicae

“…Channels displaying similar electrophysiological and pharmacological characteristics have been reported for Mpa I-like Schistocerca aLl derived homo-oligomerie receptors expressed in oocytes (Marshall et al, 1990). However, an aLl homo-oligomeric channel with much reduced nicotine sensitivity has also been reported (Amar et al, 1995), whereas Da2 (or SAD), the Drosophila homologue of aLl, demonstrated complete a-BTX insensitivity when expressed in oocytes (Sawruk et al, 1990a). In contrast, Bertrand et al (1994) could not detect functional expression of homomeric Da2 in ooeytes, indicating the possible need for additional subunits.…”

“…I and Table I) reveals substantial homology (~70%) between Mpa 1, Drosophila Da2 (also named SAD; Baumann et al, 1990;Sawruk et al, 1990a;Lansdell et al, 1997), and locust aLl (Marshall et al, 1990), and similarly between Mpa2 and the Drosophila ALS subunit (Bossy et al, 1988). As the Drosophila Da2 subunit shares 77.5% amino acid identity with its locust homologue aL I, it is likely that Mpal, with 72.2 and 69.2% sequence homology to aLl and Da2, is the comparable Myzus homologue (Table I).…”

“…melanogaster ("Drosophila") and S. gregaria (Marshall et al, 1990;GundelfInger and Hess, 1992). Initial work suggested that some insect nAChRs can give homo-oligomeric functional channels.…”

“…gregaria, generated nicotine-sensitive cation channels (Marshall et al, 1990;Amar Ct al., 1995). However, such heterologous expression studies with these subunit genes have so far shed only limited and to some extent ambiguous light on potential insect nAChR subunit compositions (Marshall et al, 1990;Sawruk et al, l990a;Bertrand et al, 1994;Amar et al, 1995;Lansdell et al, 1997).…”

A review of experimental techniques used for the heterologous expression of nicotinic acetylcholine receptors

“…In addition, Xenopus oocytes have been used successfully to examine nAChR subunits cloned from several invertebrate species, including the aphid Myzus persicae [89], the brown planthopper Nilaparvata lugens [90], the fruit fly Drosophila melanogaster [69,91], the locust Schistocerca gregaria [92] and the nematode Caenorhabditis elegans [93,94]. Frustratingly, the heterologous expression of invertebrate nAChRs has proved to be extremely difficult [59,95] and, in several instances, this has often been achieved only by co-expression with vertebrate nAChR subunits [65,91,96,97].…”