We have isolated and characterized cDNA clones encoding the murine homologue of a putative fourth Na,K-ATPase α subunit isoform (α4). The predicted polypeptide is 1032 amino acids in length and exhibits 75% amino acid sequence identity to the rat α1, α2, and α3 subunits. Within the first extracellular loop, the α4 subunit is highly divergent from other Na,K-ATPase α subunits. Because this region of Na,K-ATPase is a major determinant of ouabain sensitivity, we tested the ability of the rodent α4 subunit to transfer ouabain resistance in a transfection protocol. We find that a cDNA containing the complete rodent α4 ORF is capable of conferring low levels of ouabain resistance upon HEK 293 cells, an indication that the α4 subunit can substitute for the endogenous ouabain-sensitive α subunit of human cells. Nucleotide sequences specific for the murine α4 subunit were used to identify the chromosomal position of the α4 subunit gene. By hybridizing an α4 probe with a series of BACs, we localized the α4 subunit gene (Atp1a4) to the distal portion of mouse chromosome 1, in very close proximity to the murine Na,K-ATPase α2 subunit gene. In adult mouse tissues, we detected expression of the α4 subunit gene almost exclusively in testis, with low levels of expression in epididymis. The close similarities in the organization and expression pattern of the murine and human α4 subunit genes suggest that these two genes are orthologous. Together, our studies indicate that the α4 subunit represents a functional Na,K-ATPase α subunit isoform.
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