Five nicotinic acetylcholine receptor subunits from the Morotoge shrimp, Pandalopsis japonica: cloning, tissue distribution, and functional expression in Xenopus oocytes

The nicotinic acetylcholine receptor (nAChR) is a member of the ligand-gated ion channel (LGIC) family and is composed of five subunits arranged around a central pore. Expressed sequence tag screening and traditional cloning strategies revealed five full-length cDNAs encoding nAChR subunit homologs (Paj3, Paj10, Paj11, Paj12, and Paj1) in the Morotoge shrimp, Pandalopsis japonica. The nAChR subunits exhibited common structural characteristics, including a signal peptide sequence, a large N-terminal extracellular domain with conserved motifs for ligand binding (loops A-F), and a transmembrane (TM) domain with four hydrophobic TM motifs (TM1-TM4). Based on the conserved GEK motifs located just before TM2, all five nAChR subunits from P. japonica appear to be cation-selective ion channels. Among the five subunits, Paj3 and Paj1 clustered together with insect core groups, whereas Paj10, Paj11, and Paj12 were classified as a divergent group. Three distinct transcripts were identified in Paj3, presumably due to alternative splicing between TM3 and TM4, which may be involved in channel formation with other subunits. All five nAChR subunits were expressed predominantly in neuronal tissues, including the brain, sinus gland/X-organ complex, thoracic ganglia, and abdominal ganglia, with no significant differences in subunit expression levels among the neuronal tissues. The five shrimp nAChR subunits could not be functionally expressed in Xenopus oocytes, but coexpression of Paj1 and rat 4 subunit (R4) formed functional channels responding to acetylcholine. Functional expression of vertebrate subunit (R4) with invertebrate 1 subunit (Paj1) will expand our knowledge regarding the structural characteristics and molecular gating mechanism of invertebrate nAChRs.