Full length cDNA cloning and tissue expression of the Na+-K+-ATPase α1 subunit from Cyprinus carpio var. jian

Abstract

Na+-K+-ATPase is a ubiquitous plasma membrane enzyme that actively transports Na+ out of and K+ into animal cells.Its function is coupled to the hydrolysis of ATP and Na+-K+-ATPase plays a central role in the regu-lation of membrane potential,cell ion content,and the excitability and contractility of cells.Structurally,the Na-K-ATPase consists of two major polypeptides,a large catalytic α-subunit and a smaller glycosylated β-subunit.The α-subunit contains an intracellular ATP binding site,a phosphorylation site,and an extracellular binding site for cardiac glycosides,whereas the glycosylated β-subunit is thought to be a necessary component for maintaining stability and correct membrane orientation of the sodium pump.Four isoforms of the α-subunit and three isoforms of the β-subunit have been identified in mammals.The α1-subunit is expressed ubiquitously among tissues and is thought to play a ''housekeeping'' role in maintaining Na+ and K+ gradients and cell viability.We investigated the molecular mechanisms regulating Na+-K+-ATPase gene expression and osmoregulation in Cyprinus carpio var.jian.The full-length cDNA encoding Cyprinus carpio var.jian Na+-K+-ATPase α1 was cloned from Cyprinus car-pio var.jian using homology cloning and RACE PCR.The Na+-K+-ATPase α1 was 3 397 bp in length,including a 232 bp 5'terminal UTR,a 3 081 bp encoding region,and a 219 bp 3'terminal UTR.There are eight transmem-brane-spanning regions in the α1 subunit.Alignment of the deduced amino acid sequences of the Na+-K+-ATPase α1 and those of other vertebrates demonstrated that they shared many protein features that are common among fish and mammals.Phylogenetic analysis using MEGA 4 software revealed that the putative Na+-K+-ATPase α1 amino acid and that of Danio rerio had high similarity(95.52%).Similarly,the similarity was 92.65%,91.97%,91.38%,90.52%,90.42%,90.23%,90.22%,89.62%,and 89.51% with Chanos chanos,Rhabdosargus sarba,Sarotherodon melanotheron,Fundulus heteroclitus,Acanthopagrus schlegelii,Oncorhynchus masou,Oncorhynchus mykiss,Salmo salar,Xenopus laevis,and Homo sapiens,respectively.Real-time quantitative PCR revealed that the Na+-K+-ATPase α1 gene was expressed in all 6 tissues: brain,liver,kidney,intestines,spleen,and gills.The Na+-K+-ATPase α1 transcript was detected at a high level in the brain,at moderate levels in the gills,spleen,kid-ney,and intestines,and at low levels in the liver.