Sodium kinetics of Na,K-ATPase alpha isoforms in intact transfected HeLa cells

By participating in the regulation of ion and voltage gradients, the Na-K pump (i.e., Na,K-ATPase) influences many aspects of cellular physiology. Of the four alpha isoforms of the pump, alpha 1 is ubiquitous, alpha 2 is predominant in skeletal muscle, and alpha 3 is found in neurons and the cardiac conduction system. To determine whether the isoforms have different intracellular Na+ affinities, we used the Na+-sensitive dye sodium-binding benzofuran isophthalate (SBFI) to measure pump-mediated Na+ efflux as a function of [Na+], in human HeLa cells stably transfected with rat Na-K pump isoforms. We Na+-loaded the cells, and then monitored the time course of the decrease in [Na+](i) after removing external Na+. All transfected rat a subunits were highly ouabain resistant: the alpha 1 isoform is naturally resistant, whereas the alpha 2 and alpha 3 isoforms had been mutagenized to render them resistant. Thus, the Na+ efflux mediated by endogenous and transfected pumps could be separated by studying the cells at low (1 mu M) and high (4 mM) ouabain concentrations. We found that the apparent K-m for Na+ efflux attributable to the native human alpha 1 isoform was 12 mM, which was similar to the K-m of rat alpha 1. The alpha 2 and alpha 3 isoforms had apparent K-m's of 22 and 33 mM, respectively. The cells expressing alpha 3 had a high resting [Na+](i). The maximal activity of native alpha 1 in the alpha 3-transfected cells was only similar to 56% of native al activity in untransfected HeLa cells, suggesting that transfection with alpha 3 led to a compensatory decrease in endogenous alpha 1 pumps. We conclude that the apparent K-m(Na+) for rat Na-K pump isoforms increases in the sequence alpha 1 < alpha 2 < alpha 3. The alpha 3 isoform may be suited for handling large Na+ loads in electrically active cells.