Na,K-ATPase plays a crucial role in cellular ion homeostasis and is the pharmacological receptor for digitalis in man. Nine different human Na,K-ATPase isozymes, composed of 3 ␣ and ␤ isoforms, were expressed in Xenopus oocytes and were analyzed for their transport and pharmacological properties. According to ouabain binding and K ؉ -activated pump current measurements, all human isozymes are functional but differ in their turnover rates depending on the ␣ isoform. On the other hand, variations in external K ؉ activation are determined by a cooperative interaction mechanism between ␣ and ␤ isoforms with ␣2-␤2 complexes having the lowest apparent K ؉ affinity. ␣ Isoforms influence the apparent internal Na ؉ affinity in the order ␣1 > ␣2 > ␣3 and the voltage dependence in the order ␣2 > ␣1 > ␣3. All human Na,K-ATPase isozymes have a similar, high affinity for ouabain. However, ␣2-␤ isozymes exhibit more rapid ouabain association as well as dissociation rate constants than ␣1-␤ and ␣3-␤ isozymes. Finally, isoformspecific differences exist in the K ؉ /ouabain antagonism which may protect ␣1 but not ␣2 or ␣3 from digitalis inhibition at physiological K ؉ levels. In conclusion, our study reveals several new functional characteristics of human Na,K-ATPase isozymes which help to better understand their role in ion homeostasis in different tissues and in digitalis action and toxicity.

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