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.
Voltage-dependent calcium (Ca2+) channels are involved in many specialized cellular functions, and are controlled by intracellular signals such as heterotrimeric G-proteins, protein kinases and calmodulin (CaM). However, the direct role of small G-proteins in the regulation of Ca2+ channels is unclear. We report here that the GTP-bound form of kir/Gem, identified originally as a Ras-related small G-protein that binds CaM, inhibits high-voltage-activated Ca2+ channel activities by interacting directly with the beta-subunit. The reduced channel activities are due to a decrease in alpha1-subunit expression at the plasma membrane. The binding of Ca2+/CaM to kir/Gem is required for this inhibitory effect by promoting the cytoplasmic localization of kir/Gem. Inhibition of L-type Ca2+ channels by kir/Gem prevents Ca2+-triggered exocytosis in hormone-secreting cells. We propose that the small G-protein kir/Gem, interacting with beta-subunits, regulates Ca2+ channel expression at the cell surface.
FXYD proteins belong to a family of small-membrane proteins. Recent experimental evidence suggests that at least five of the seven members of this family, FXYD1 (phospholemman), FXYD2 (gamma-subunit of Na-K-ATPase), FXYD3 (Mat-8), FXYD4 (CHIF), and FXYD7, are auxiliary subunits of Na-K-ATPase and regulate Na-K-ATPase activity in a tissue- and isoform-specific way. These results highlight the complexity of the regulation of Na+ and K+ handling by Na-K-ATPase, which is necessary to ensure appropriate tissue functions such as renal Na+ reabsorption, muscle contractility, and neuronal excitability. Moreover, a mutation in FXYD2 has been linked to cases of human hypomagnesemia, indicating that perturbations in the regulation of Na-K-ATPase by FXYD proteins may be critically involved in pathophysiological states. A better understanding of this novel regulatory mechanism of Na-K-ATPase should help in learning more about its role in pathophysiological states. This review summarizes the present knowledge of the role of FXYD proteins in the modulation of Na-K-ATPase as well as of other proteins, their regulation, and their structure-function relationship.
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.
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