Molecular cloning and characterization of porcine Na⁺/K⁺-ATPase isoform α4.

Na/K-ATPase is responsible for maintaining electrochemical gradients of Na and K, which is essential for a variety of cellular functions including neuronal activity. The α-subunit of the Na/K-ATPase is composed of four different polypeptides (α1-α4) encoded by different genes. Na,K-ATPase α4, encoded by the ATP1A4 gene, is expressed in testis and in male germ cells of humans, rats and mice. The α4 polypeptide has an important role in sperm motility, and is essential for male fertility. Here we present the RT-PCR cloning and characterization of the porcine ATP1A4 cDNA coding for Na⁺/K⁺-ATPase polypeptide α4. The Na⁺/K⁺-ATPase polypeptide α4, consisting of 1030 amino acids, displays a high homology with its human counterpart (86%). Phylogenetic analysis demonstrated that porcine Na⁺/K⁺-ATPase polypeptide α4 is closely related to other mammalian counterparts. In addition, the genomic structure of the porcine ATP1A4 gene was determined, and the intron-exon organization was found to be similar to that of the human ATP1A4 gene. The promoter sequence for the porcine ATP1A4 gene was also identified. Investigation of the genetic variation in the porcine ATP1A4 gene revealed a missense A/G SNP in exon 18. This A/G polymorphism results in a substitution of a methionine to a glycine residue (M888G). A very high overall DNA methylation rate of the ATP1A4 gene, 70-80%, was observed in both brain and liver. Expression analysis demonstrated that the porcine ATP1A4 gene is predominantly expressed in testis. The sequence of the porcine ATP1A4 cDNA encoding the Na⁺/K⁺-ATPase α4 protein has been submitted to GenBank under the accession number GenBank Accession No. MG587082.