EXPRESSION OF NA+-K+-ATPASE ALPHA-SUBUNIT AND BETA-SUBUNIT ALONG RAT NEPHRON - ISOFORM SPECIFICITY AND RESPONSE TO HYPOKALEMIA

The activity of Na+-K+-adenosinetriphosphatase (Na+-K+-ATPase), the sodium pump, which drives active Na+ reabsorption along the nephron, varies over an order of magnitude, depending on the nephron segment, and activity is increased in the outer medullary collecting tubule (MCT) during hypokalemia. The aims of the present study were to assess abundance of sodium pump alpha(1)- and beta(1)-subunits in dissected nephron segments of the rat by immunoblotting, to determine if alpha(2)- or alpha(3)-protein could be detected in the collecting tubules, as suggested by Barlet-Bas et al. (C. Barlet-Bas, E. Arystarkhova, L. Cheval, S. Marsy, K. Sweadner, N. Modyanov, and A. Doucet. J. Biol. Chem. 268: 11512-11515, 1993) for rabbit, and to determine if alpha(1) and beta(1) were increased in MCT by hypokalemia. Tubules from the rat were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (12-100 mm/lane), blotted, and probed with subunit-specific antisera. alpha(1) and beta(1), detected in all tubule segments assayed, were highest in cortical and medullary thick ascending limbs and proximal convoluted tubule (PCT), lower in the MCT and barely detectable in proximal straight tubule. In the cortical collecting tubule (CCT), alpha(1) abundance was equivalent to that in PCT, whereas beta(1) and enzymatic activity were both less than one-half of that in PCT. After 2 wk of a K+-deficient diet, alpha(1)- and beta(1)-subunit levels in MCT increased 3.4 +/- 0.6- and 11.7 +/- 4.0-fold, respectively, associated with a 5-fold increase in activity. alpha(2) and alpha(3) were not detected in the CCT or MCT. In conclusion, Na+-K+-ATPase subunit levels can be monitored at the protein level in dissected tubules, alpha(1)- and beta(1)-isoform subunits were detected in all tubules assayed, whereas alpha(2) and alpha(3) could not be detected, and the increases in Na+-K+-ATPase activity in rat MCT from hypokalemic rat can be accounted for by increases in pool sizes of alpha(1)- and beta(1)-subunits.