Cloning and Expression Study of Five Nicotinic Acetylcholine Receptor Subunits from the Morotoge shrimp (Pandalopsis japonica)

Abstract

The Nicotinic Acetylcholine Receptor (nAChR) is a family member of ligand-gated ion channels (LGICs), which is functional with five subunits forming a central pore. As result of EST screening and traditional cloning strategy, total 5 full length cDNAs encoding nAChR subunit homologs (Pajα4, Pajα10, Pajα11, Pajα12 and Pajβ1) were identified from Pandalopsis japonica. Obtained nAChR subunits from P. japonica exhibited the common structural characteristics including the signal peptide sequence, the a large N-terminal extracellular domain with conserved motifs (loop A-F), and the transmembrane domain with four hydrophobic transmembrane motifs (TM1~4). Based on the conserved GEK motifs right ahead of the TM2, all five nAChR subunits from P. japonica appear to be the cation-selective ion channel. Among 5 subunits, Pajα4 and Pajβ1 were clustered together with insect core group, whereas Pajα10, Pajα11, and Pajα12 were classified as divergent group. Three distinct transcripts were identified in Pajα4 presumably by the alternative splicing. All 5 nAChR subunits were predominantly expressed in neuronal tissues including brain, sinus gland/X-organ complex (SG/XO), thoracic ganglia, and abdominal ganglia. Each subunit was expressed without any significant difference in expression level throughout the neuronal tissues. Functional expression of 5 shrimp nAChR subunits failed by themselves in Xenopus oocytes, however, coexpression with Pajβ1 and rat α4 subunit (Rα4) formed functional channel responding to acetylcholine (ACh). Functional expression of vertebrate α subunit (Rα4) with invertebrate β1 subunit (Pajβ1) would help us expand our knowledge about structural character and molecular gating mechanism of invertebrate nAChRs.