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.

A family of small, single-span membrane proteins (the FXYD family) has recently been defined based on their sequence and structural homology. Some members of this family have already been identified as tissue-specific regulators of Na,K-ATPase (NKA). In the present study, we demonstrate that phospholemman (PLM) (FXYD1), so far considered to be a heart-and muscle-specific channel or channel-regulating protein, associates specifically and stably with six different ␣-␤ isozymes of NKA after coexpression in Xenopus oocytes, and with ␣1-␤, and less efficiently with ␣2-␤ isozymes, in native cardiac and skeletal muscles. Stoichiometric association of PLM with NKA occurs posttranslationally either in the Golgi or the plasma membrane. Interaction of PLM with NKA induces a small decrease in the external K ؉ affinity of ␣1-␤1 and ␣2-␤1 isozymes and a nearly 2-fold decrease in the internal Na ؉ affinity. In conclusion, this study demonstrates that PLM is a tissue-specific regulator of NKA that may play an essential role in muscle contractility.

P.Be Âguin and G.Crambert contributed equally to this workRecently, corticosteroid hormone-induced factor (CHIF) and the g-subunit, two members of the FXYD family of small proteins, have been identi®ed as regulators of renal Na,K-ATPase. In this study, we have investigated the tissue distribution and the structural and functional properties of FXYD7, another family member which has not yet been characterized. Expressed exclusively in the brain, FXYD7 is a type I membrane protein bearing N-terminal, post-translationally added modi®cations on threonine residues, most probably O-glycosylations that are important for protein stabilization. Expressed in Xenopus oocytes, FXYD7 can interact with Na,K-ATPase a1±b1, a2±b1 and a3±b1 but not with a±b2 isozymes, whereas, in brain, it is only associated with a1±b isozymes. FXYD7 decreases the apparent K + af®nity of a1±b1 and a2±b1, but not of a3±b1 isozymes. These data suggest that FXYD7 is a novel, tissue-and isoformspeci®c Na,K-ATPase regulator which could play an important role in neuronal excitability.