Embryonic Expression of the Putative γ Subunit of the Sodium Pump Is Required for Acquisition of Fluid Transport Capacity during Mouse Blastocyst Development

Abstract

The sodium/potassium pump, Na+,K+-ATPase, is generally understood to function as a heterodimer of two subunits, a catalytic α subunit and a noncatalytic, glycosylated β subunit. Recently, a putative third subunit, the γ subunit, was cloned. This small protein (6.5 kD) coimmunoprecipitates with the α and β subunits and is closely associated with the ouabain binding site on the holoenzyme, but its function is unknown. We have investigated the expression of the γ subunit in preimplantation mouse development, where Na+,K+-ATPase plays a critical role as the driving force for blastocoel formation (cavitation). Using reverse transcriptase-polymerase chain reaction, we demonstrated that the γ subunit mRNA accumulates continuously from the eight-cell stage onward and that it cosediments with polyribosomes from its time of first appearance. Confocal immunofluorescence microscopy revealed that the γ subunit itself accumulates and is localized at the blastomere surfaces up to the blastocyst stage. In contrast with the α and β subunits, the γ subunit is not concentrated in the basolateral surface of the polarized trophectoderm layer, but is strongly expressed at the apical surface as well. When embryos were treated with antisense oligodeoxynucleotide complementary to the γ subunit mRNA, ouabain-sensitive K+ transport (as indicated by 86Rb+ uptake) was reduced and cavitation delayed. However, Na+,K+-ATPase enzymatic activity was unaffected as determined by a direct phosphorylation assay (“back door” phosphorylation) applied to plasma membrane preparations. These results indicate that the γ subunit, although not an integral component of Na+,K+-ATPase, is an important determinant of active cation transport and that, as such, its embryonic expression is essential for blastocoel formation in the mouse.