The Na,K-ATPase alpha 4 gene (Atp1a4) encodes a ouabain-resistant alpha subunit and is tightly linked to the alpha 2 gene (Atp1a2) on mouse chromosome 1

We have isolated and characterized cDNA clones encoding the murine homologu e of a putative fourth Na,K-ATPase alpha subunit isoform (alpha 4). The pre dicted polypeptide is 1032 amino acids in length and exhibits 75% amino aci d sequence identity to the rat alpha 1, alpha 2, and alpha 3 subunits. With in the first extracellular loop, the alpha 4 subunit is highly divergent fr om other Na,K-ATPase alpha subunits. Because this region of Na,K-ATPase is a major determinant of ouabain sensitivity, we tested the ability of the ro dent alpha 4 subunit to transfer ouabain resistance in a transfection proto col. We find that a cDNA containing the complete rodent alpha 4 ORF is capa ble of conferring low levels of ouabain resistance upon HEK 293 cells, an i ndication that the alpha 4 subunit can substitute for the endogenous ouabai n-sensitive a subunit of human cells. Nucleotide sequences specific for the murine alpha 4 subunit were used to identify the chromosomal position of t he alpha 4 subunit gene. By hybridizing an alpha 4 probe with a series of B ACs, we localized the alpha 4 subunit gene (Atp1a4) to the distal portion o f mouse chromosome 1, in very close proximity to the murine Na,K-ATPase alp ha 2 subunit gene, In adult mouse tissues, we detected expression of the al pha 4 subunit gene almost exclusively in testis, with low levels of express ion in epididymis. The close similarities in the organization and expressio n pattern of the murine and human alpha 4 subunit genes suggest that these two genes are orthologous. Together, our studies indicate that the alpha 4 subunit represents a functional Na,K-ATPase alpha subunit isoform.