In the present study the alternative splicing of
In the present study, the alternative splicing of several nAChR subunits was identified in the oriental migratory locust (Locusta migratoria manilensis Meyen), a serious worldwide pest to agricultural production and also a model insect species . Several α subunits were tried to co-express with rat β2 subunit and the influences of subunit alternative splicing on Lonafarnib potencies were tested on some successfully constructed nAChRs in Xenopus oocytes.
Materials and methods
Discussion Nicotinic acetylcholine receptors (nAChRs), as a prototypical member of Cys-loop receptors, are comprised of five subunits into a functional unit. When five copies of one subunit construct a receptor, it is called as a homopentamer. In contrast, the receptors are commonly assembled from different types of subunits and called as a heteropentamer , . A dozen subunits in one insect species provide the high diversity of insect nAChRs with distinct functional and pharmacological properties , . However, even in the case of insects such as Drosophila with all subunits identified, considerable problems remain in generating functional insect nAChRs in heterologous expression systems , . Even through co-expression with vertebrate subunits, such as the rat β2 subunit, only few hybrid nAChRs were successfully constructed with insect α subunits, such as α1 and α2 from Myzus Persicae and L. migratoria, , and α1, α2 and α3 from D. melanogaster, , , and α1, α2 and α8 from Nilaparvata lugens, , , and so on which were not listed here. In the present study, seven functional hybrid receptors containing rat β2 subunit and one L. migratoria α subunit were constructed in Xenopus oocytes. The recombinant receptors containing rat β2 subunit and one of insect α4, α5 and α9 subunits were first reported here. On hybrid nAChRs containing N. lugens α8 and rat β2 subunits, acetylcholine showed very weak agonist potency with very small Imax value (9.7±2.1nA) and big EC50 value (95.8±11.7μM) . In this study, the hybrid receptor containing Locα8 and rat β2 subunits showed the highest sensitivity to acetylcholine (EC50=18.5±3.6μM) among all hybrid nAChRs, although the Imax value was still small (29.6±4.7nA). In insects, as well as in vertebrates, alternative splicing in subunits would increase the diversity of nAChRs in function and pharmacology. The ligand-binding site is located at the interface between two subunits, with amino acid residues contributed by three distinct loops of α subunits (referred to as binding site loops A, B, and C) and three loops of non-α-subunits (loops D, E, and F) , . Changes in amino sequences within and at the intervals of these specific loops often varied some key properties of recombinant receptors, such as sensitivities to acetylcholine and neonicotinoid insecticides , , , , , . So, alternative splicing in insect subunits would be able to comprise more nAChRs in insects with distinct functional and pharmacological properties, in native nAChRs as well as recombinant hybrid receptors , , . In this study, alternative splicing at the region from loop A to the N-terminus (missing conserved residues in Loop D) in Locα1 and Locα9 resulted in the increase of acetylcholine sensitivities on the hybrid receptors Locα1/β2 and Locα9/β2. Although the cytoplasmic loop between TM3 and TM4 was far from the ligand-binding site located extracellularly, alternative splicing, a 34-residue insertion, in Locα8 cytoplasmic loop caused a significant increase in Imax value of acetylcholine on Locα8/β2. The results indicated that the alternative splicing at cytoplasmic regions, as well as the splicing at or close to the ligand-binding site, would change the agonist potencies on receptors. In conclusion, the alternative splicing was identified in six α subunits from L. migratoria. By heterologous expression of α subunits with rat β2 subunit in Xenopus oocytes, the influences of alternative splicing on functional properties were tested, which showed that some alternative splicing could significantly change the agonist potency of acetylcholine on hybrid receptors. The results revealed that alternative splicing in insect nAChR subunits would increase the diversity of nAChRs. More properties in function and pharmacology will be tested in further studies, such as sensitivities to neonicotinoid insecticides and nAChRs specific antagonists.