Change in gene functions (gene cooption) is one of the key mechanisms of molecular evolution. Genes can acquire new functions via alteration in properties of encoded proteins and/or via changes in temporal or spatial regulation of expression. Here we demonstrate radical changes in the functions of orthologous ATP1B4 genes during evolution of vertebrates. Expression of ATP1B4 genes is brain-specific in teleost fishes, whereas it is predominantly muscle-specific in tetrapods. The encoded ␤m-proteins in fish, amphibian, and avian species are ␤-subunits of Na,K-ATPase located in the plasma membrane. In placental mammals ␤m-proteins lost their ancestral functions, accumulate in nuclear membrane of perinatal myocytes, and associate with transcriptional coregulator Ski-interacting protein (SKIP). Through interaction with SKIP, eutherian ␤m acquired new functions as exemplified by regulation of TGF-␤-responsive reporters and by augmentation of mRNA levels of Smad7, an inhibitor of TGF-␤ signaling. Thus, orthologous vertebrate ATP1B4 genes represent an instance of gene cooption that created fundamental changes in the functional properties of the encoded proteins.ATP1B4 ͉ gene cooption ͉ skeletal muscle development ͉ TGF-␤ ͉ Smad7

The structural organization of nongastric H-K-ATPase, unlike that of closely related Na-K-ATPase and gastric H-K-ATPase, is not well characterized. Recently, we demonstrated that nongastric H-K-ATPase α-subunit (αng) is expressed in apical membranes of rodent prostate. Its highest level, as well as relative abundance, with respect to α1-isoform of Na-K-ATPase, was observed in anterior lobe. Here, we aimed to determine the subunit composition of nongastric H-K-ATPase through the detailed analysis of the expression of all known X-K-ATPase β-subunits in rat anterior prostate (AP). RT-PCR detects transcripts of β-subunits of Na-K-ATPase only. Measurement of absolute protein content of these three β-subunit isoforms, with the use of quantitative Western blotting of AP membrane proteins, indicates that the abundance order is β1 > β3 ≫ β2. Immunohistochemical experiments demonstrate that β1 is present predominantly in apical membranes, coinciding with αng, whereas β3 is localized in the basolateral compartment, coinciding with α1. This is the first direct demonstration of the αng-β1 colocalization in situ indicating that, in rat AP, αng associates only with β1. The existence of αng-β1 complex has been confirmed by immunoprecipitation experiments. These results indicate that β1-isoform functions as the authentic subunit of Na-K-ATPase and nongastric H-K-ATPase. Putatively, the intracellular polarization of X-K-ATPase isoforms depends on interaction with other proteins.

Cardiac steroids inhibit Na,K-ATPase and the related non-gastric H,K-ATPase, while they do not interact with gastric H,K-ATPase. Introducing an arginine, the residue present in the gastric H,K-ATPase, in the second extracellular loop at the corresponding position 334 in the human non-gastric H,K-ATPase (D334R mutation) rendered it completely resistant to 2mM ouabain. The corresponding mutation (E319R) in alpha1 Na,K-ATPase produced a approximately 2-fold increase of the ouabain IC(50) in the ouabain-resistant rat alpha1 Na,K-ATPase and a large decrease of the ouabain affinity of human alpha1 Na,K-ATPase, on the other hand this mutation had no effect on the affinity for the aglycone ouabagenin. These results provide a strong support for the orientation of ouabain in its biding site with its sugar moiety interacting directly with the second extracellular loop.