Functional Modulation of the Sodium Pump: The Regulatory Proteins “Fixit”

Self Cite

In Na,K-ATPase membrane preparations from shark rectal glands, we have previously identified an FXYD domain-containing protein, phospholemman-like protein from shark, PLMS. This protein was shown to associate and modulate shark Na,K-ATPase activity in vitro. Here we describe the complete coding sequence, expression, and cellular localization of PLMS in the rectal gland of the shark Squalus acanthias. The mature protein contained 74 amino acids, including the N-terminal FXYD motif and a C-terminal protein kinase multisite phosphorylation motif. The sequence is preceded by a 20 amino acid candidate cleavable signal sequence. Immunogold labeling of the Na,K-ATPase ␣-subunit and PLMS showed the presence of ␣ and PLMS in the basolateral membranes of the rectal gland cells and suggested their partial colocalization. Furthermore, through controlled proteolysis, the C terminus of PLMS containing the protein kinase phosphorylation domain can be specifically cleaved. Removal of this domain resulted in stimulation of maximal Na,K-ATPase activity, as well as several partial reactions. Both the E 1 ϳP 3 E 2 -P reaction, which is partially rate-limiting in shark, and the K ؉ deocclusion reaction, E 2 (K) 3 E 1 , are accelerated. The latter may explain the finding that the apparent Na ؉ affinity was increased by the specific C-terminal PLMS truncation. Thus, these data are consistent with a model where interaction of the phosphorylation domain of PLMS with the Na,K-ATPase ␣-subunit is important for the modulation of shark Na,K-ATPase activity.

Section: Discussionmentioning

confidence: 77%

Section: Discussionmentioning

confidence: 84%

Regulation of Na,K-ATPase by PLMS, the Phospholemman-like Protein from Shark

Mahmmoud

Cramb

Maunsbach

et al. 2003

Self Cite

“…As could be expected for a protein with such a central physiological role, Na ϩ ,K ϩ -ATPase is closely regulated at several levels. A unique mode of regulation of the Na ϩ ,K ϩ -ATPase has been described recently, involving interactions between the ␣/␤ complex and the FXYD proteins (1)(2)(3)(4). The FXYD family consists of seven short single-span trans-membrane proteins (extracellular N terminus), named after the conserved FXYD motif in the extracellular segment (5).…”

mentioning

confidence: 99%

“…In kidney ␥ is expressed as two splice variants, ␥a and ␥b (5,6). Six members of the family, FXYD1 (PLM), FXYD2 (␥), FXYD3 (Mat-8), FXYD4 (CHIF), FXYD5 (related to ion channel), and FXYD7, are now known to interact specifically with the Na ϩ ,K ϩ -ATPase and to alter the pump kinetics in characteristic and different ways (2)(3)(4). Thus, the working hypothesis is that FXYD proteins function as tissue-specific modulators of Na ϩ ,K ϩ -ATPase, which adjust its kinetic behavior to the special needs of the given tissue, cell type, or physiological state (3,4,7,8).…”

mentioning

confidence: 99%

Structural Interactions between FXYD Proteins and Na+,K+-ATPase

Lindzen

Gottschalk²,

Füzesi

et al. 2006

“…In the last few years evidence for a completely new mechanism of regulation of Na,K-ATPase has emerged; the intrinsic properties of Na,K-ATPase can be modulated by association with a small single span membrane protein belonging to the "FXYD" family (3). Expression of the FXYD proteins is abundant in tissues that are involved in fluid and solute transport or that are electrically excitable (4,5).…”

mentioning

confidence: 99%

Stress-induced Expression of the γ Subunit (FXYD2) Modulates Na,K-ATPase Activity and Cell Growth

Wetzel

Pascoa

Arystarkhova

2004

In kidney, the Na,K-ATPase is associated with a single span protein, the ␥ subunit (FXYD2). Two splice variants are differentially expressed along the nephron and have a differential influence on Na,K-ATPase when stably expressed in mammalian cells in culture. Here we used a combination of gene induction and gene silencing techniques to test the functional impact of ␥ by means other than transfection. NRK-52E cells (of proximal tubule origin) do not express ␥ as a protein under regular tissue culture conditions. However, when they were exposed to hyperosmotic medium, induction of only the ␥a splice variant was observed, which was accompanied by a reduction in the rate of cell division. Kinetic analysis of stable enzyme properties from control (␣1␤1) and hypertonicity-treated cultures (␣1␤1␥a) revealed a significant reduction (up to 60%) of Na,K-ATPase activity measured under V max conditions with little or no change in the amounts of ␣1␤1. This effect as well as the reduction in cell growth rate was practically abolished when ␥ expression was knocked down using specific small interfering RNA duplexes. Surprisingly, a similar induction of endogenous ␥a because of hypertonicity was seen in rat cell lines of other than renal origin: C6 (glioma), PC12 (pheochromocytoma), and L6 (myoblasts). Furthermore, exposure of NRK-52E cells to other stress inducers such as heat shock, exogenous oxidation, and chemical stress also resulted in a selective induction of ␥a. Taken together, the data imply that induction of ␥a may have adaptive value by being a part of a general cellular response to genotoxic stress.