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      Phospholemman (FXYD1) associates with Na,K-ATPase and regulates its transport properties

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          Abstract

          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 alpha-beta isozymes of NKA after coexpression in Xenopus oocytes, and with alpha1-beta, and less efficiently with alpha2-beta 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 alpha1-beta1 and alpha2-beta1 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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          Most cited references 33

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          Efficient in vitro synthesis of biologically active RNA and RNA hybridization probes from plasmids containing a bacteriophage SP6 promoter.

           M. Green,  T Maniatis,  K Zinn (1984)
          A simple and efficient method for synthesizing pure single stranded RNAs of virtually any structure is described. This in vitro transcription system is based on the unusually specific RNA synthesis by bacteriophage SP6 RNA polymerase which initiates transcription exclusively at an SP6 promoter. We have constructed convenient cloning vectors that contain an SP6 promoter immediately upstream from a polylinker sequence. Using these SP6 vectors, optimal conditions have been established for in vitro RNA synthesis. The advantages and uses of SP6 derived RNAs as probes for nucleic acid blot and solution hybridizations are demonstrated. We show that single stranded RNA probes of a high specific activity are easy to prepare and can significantly increase the sensitivity of nucleic acid hybridization methods. Furthermore, the SP6 transcription system can be used to prepare RNA substrates for studies on RNA processing (1,5,9) and translation (see accompanying paper).
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            Transport and pharmacological properties of nine different human Na, K-ATPase isozymes.

            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 alpha and beta 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 alpha isoform. On the other hand, variations in external K(+) activation are determined by a cooperative interaction mechanism between alpha and beta isoforms with alpha2-beta2 complexes having the lowest apparent K(+) affinity. alpha Isoforms influence the apparent internal Na(+) affinity in the order alpha1 > alpha2 > alpha3 and the voltage dependence in the order alpha2 > alpha1 > alpha3. All human Na,K-ATPase isozymes have a similar, high affinity for ouabain. However, alpha2-beta isozymes exhibit more rapid ouabain association as well as dissociation rate constants than alpha1-beta and alpha3-beta isozymes. Finally, isoform-specific differences exist in the K(+)/ouabain antagonism which may protect alpha1 but not alpha2 or alpha3 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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              The gamma subunit is a specific component of the Na,K-ATPase and modulates its transport function.

              The role of small, hydrophobic peptides that are associated with ion pumps or channels is still poorly understood. By using the Xenopus oocyte as an expression system, we have characterized the structural and functional properties of the gamma peptide which co-purifies with Na,K-ATPase. Immuno-radiolabeling of epitope-tagged gamma subunits in intact oocytes and protease protection assays show that the gamma peptide is a type I membrane protein lacking a signal sequence and exposing the N-terminus to the extracytoplasmic side. Co-expression of the rat or Xenopus gamma subunit with various proteins in the oocyte reveals that it specifically associates only with isozymes of Na,K-ATPase. The gamma peptide does not influence the formation and cell surface expression of functional Na,K-ATPase alpha-beta complexes. On the other hand, the gamma peptide itself needs association with Na,K-ATPase in order to be stably expressed in the oocyte and to be transported efficiently to the plasma membrane. Gamma subunits do not associate with individual alpha or beta subunits but only interact with assembled, transport-competent alpha-beta complexes. Finally, electrophysiological measurements indicate that the gamma peptide modulates the K+ activation of Na,K pumps. These data document for the first time the membrane topology, the specificity of association and a potential functional role for the gamma subunit of Na,K-ATPase.
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                Author and article information

                Journal
                Proceedings of the National Academy of Sciences
                Proceedings of the National Academy of Sciences
                Proceedings of the National Academy of Sciences
                0027-8424
                1091-6490
                August 20 2002
                August 08 2002
                August 20 2002
                : 99
                : 17
                : 11476-11481
                Article
                10.1073/pnas.182267299
                123281
                12169672
                801f61b8-dd62-4a1c-aa5d-e7004284079d
                © 2002
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