FXYD Proteins Research Articles

FXYD7, the first brain- and isoform-specific regulator of Na,K-ATPase: biosynthesis and function of its posttranslational modifications.

The FXYD protein family has recently been defined as a result of the search for homologues of the Na,K-ATPase gamma subunit, CHIF, and phospholemman in EST and gene data banks. FXYD7 has been seen to have a role as a brain- and isozyme-specific regulator of Na/K-ATPase. In this study, the biosynthesis, membrane topology, nature, and role of the processing of FXYD7 are investigated.

Themes in ion pump regulation.

The Na,K-ATPase is an ion pump present in the plasma membrane. It is of vital importance for cell and body homeostasis and as such is under strict hormonal control. The molecular basis for the acute regulation of Na,K-ATPase is multisite phosphorylation by protein kinases that can alter its behavior. This includes direct effects on the Na,K-ATPase activity, regulation by membrane trafficking, and even dynamic regulation of interaction with regulatory proteins. In shark Na,K-ATPase, the latter includes functional interaction with a small hydrophobic protein of the FXYD protein family, phospholemman-like protein from shark, PLMS. This article summarizes our recent work on the mechanisms involved in the acute regulation of the Na,K-ATPase studied using a plasma membrane preparation from shark salt glands as an epithelial transport model.

FXYD Proteins: New Tissue-Specific Regulators of the Ubiquitous Na,K-ATPase

Maintenance of the Na+ and K+ gradients between the intracellular and extracellular milieus of animal cells is a prerequisite for basic cellular homeostasis and for functions of specialized tissues. The Na,K-ATPase, an oligomeric P-type adenosine triphosphatase (ATPase), is composed of a catalytic α subunit and a regulatory β subunit and is the main player that fulfils these tasks. A variety of regulatory mechanisms are necessary to guarantee appropriate Na,K-ATPase expression and activity adapted to changing physiological demands. Recently, a regulatory mechanism was defined that is mediated by interaction of Na,K-ATPase with small proteins of the FXYD family, which possess a single transmembrane domain and so far have been considered as channels or regulators of ion channels. The mammalian FXYD proteins FXYD1 through FXYD7 exhibit tissue-specific distribution. Phospholemman (FXYD1) in heart and skeletal muscle, the γ subunit of Na,K-ATPase (FXYD2) and corticosteroid hormone-induced factor (FXYD4, also known as CHIF) in the kidney, and FXYD7 in the brain associate preferentially with the widely expressed Na,K-ATPase α1-β1 isozyme and modulate its transport activity in a way that conforms to tissue-specific requirements. Thus, tissue- and isozyme-specific interaction of Na,K-ATPase with FXYD proteins contributes to proper handling of Na+ and K+ by the Na,K-ATPase, and ensures correct function in such processes as renal Na+-reabsorption, muscle contraction, and neuronal excitability.

FXYD proteins: new tissue-specific regulators of the ubiquitous Na,K-ATPase.

Maintenance of the Na+ and K+ gradients between the intracellular and extracellular milieus of animal cells is a prerequisite for basic cellular homeostasis and for functions of specialized tissues. The Na,K-ATPase, an oligomeric P-type adenosine triphosphatase (ATPase), is composed of a catalytic alpha subunit and a regulatory beta subunit and is the main player that fulfils these tasks. A variety of regulatory mechanisms are necessary to guarantee appropriate Na,K-ATPase expression and activity adapted to changing physiological demands. Recently, a regulatory mechanism was defined that is mediated by interaction of Na,K-ATPase with small proteins of the FXYD family, which possess a single transmembrane domain and so far have been considered as channels or regulators of ion channels. The mammalian FXYD proteins FXYD1 through FXYD7 exhibit tissue-specific distribution. Phospholemman (FXYD1) in heart and skeletal muscle, the gamma subunit of Na,K-ATPase (FXYD2) and corticosteroid hormone-induced factor (FXYD4, also known as CHIF) in the kidney, and FXYD7 in the brain associate preferentially with the widely expressed Na,K-ATPase alpha1-beta1 isozyme and modulate its transport activity in a way that conforms to tissue-specific requirements. Thus, tissue- and isozyme-specific interaction of Na,K-ATPase with FXYD proteins contributes to proper handling of Na+ and K+ by the Na,K-ATPase, and ensures correct function in such processes as renal Na+-reabsorption, muscle contraction, and neuronal excitability.

Expression and characterization of the FXYD ion transport regulators for NMR structural studies in lipid micelles and lipid bilayers

The proteins PLM (phospholemman), CHIF (channel inducing factor), and Mat8 (mammary tumor protein 8 kDa) are members of the FXYD family of ion transport regulatory membrane proteins. Here we describe their cloning and expression in Escherichia coli, and their purification for NMR structural studies in lipid micelles and lipid bilayers. The molecular masses of the purified recombinant FXYD proteins, determined from SDS-PAGE and from MALDI TOF mass spectrometry, reflect monomeric species. The solution NMR and CD spectra in SDS micelles show that they adopt helical conformations. The solid-state NMR spectra in lipid bilayers give the first view of their transmembrane architecture.

A functional interaction between CHIF and Na-K-ATPase: implication for regulation by FXYD proteins.

Like the gamma-subunit of Na-K-ATPase, the corticosteroid hormone-induced factor (CHIF) is a member of the FXYD family of one-transmembrane-segment proteins. Both CHIF and two splice variants of gamma, gamma(a) and gamma(b), are expressed in the kidney. Immunolocalization experiments demonstrate mutually exclusive expression of CHIF and gamma in different nephron segments. Specific coimmunoprecipitation experiments demonstrate the existence in kidney membranes of the complexes alpha/beta/gamma(a), alpha/beta/gamma(b), and alpha/beta/CHIF and exclude mixed complexes such as alpha/beta/gamma(a)/gamma(b) and alpha/beta/gamma/CHIF. CHIF has been expressed in HeLa cells harboring the rat alpha(1)-subunit of Na-K-ATPase. (86)Rb flux experiments demonstrate that CHIF induces a two- to threefold increase in apparent affinity for cytoplasmic Na (K'(Na)) but does not affect affinity for extracellular K (Rb) ions (K'(K)) or V(max). Measurements of Na-K-ATPase using isolated membranes show similar but smaller effects of CHIF on K'(Na), whereas K'(K) and K'(ATP) are unaffected. The functional effects of CHIF differ from those of gamma. An implication of these findings is that other FXYD proteins could act as tissue-specific modulators of Na-K-ATPase.

Brain Modulator of Na,K-ATPase

The Na,K-ATPases (adenosine triphosphatases) are regulated by interactions with a family of membrane proteins known as FXYD proteins, so named because of a shared sequence motif. Béguin et al. characterized a new member of the family called FXYD7. Of the rat tissues sampled, FXYD was expressed only in brain and was present in both neurons and glial cells. Like other FXYD proteins, FXYD7 interacted with the Na,K-ATPase and could alter the function of that enzyme. Na,K-ATPases are dimers composed of combinations of four α and three β isoforms. FXYD7 appeared to interact only with α1-β1 isozymes. Studies in injected oocytes showed that unlike other FXYD proteins, such as CHIF (corticosteroid hormone-induced factor), FXYD7 did not alter activation of the Na,K-ATPase by Na+. Rather, FXYD7 decreased the apparent affinity of the Na,K-ATPase for K+. The authors suggest that such modulation of the affinity of the Na,K-ATPase for K+ might be important for response to high concentrations of K+ that occur after intense neuronal activity.P. Béguin, G. Crambert, F. Monnet-Tschudi, M. Uldry, J.-D. Horisberger, H. Garty, K. Geering, FXYD7 is a brain-specific regulator of Na,K-ATPaseα1-ß isozymes. EMBO J. 21, 3264-3273 (2002). [Abstract] [Full Text]

Brain Modulator of Na,K-ATPase

The Na,K-ATPases (adenosine triphosphatases) are regulated by interactions with a family of membrane proteins known as FXYD proteins, so named because of a shared sequence motif. Béguin et al. characterized a new member of the family called FXYD7. Of the rat tissues sampled, FXYD was expressed only in brain and was present in both neurons and glial cells. Like other FXYD proteins, FXYD7 interacted with the Na,K-ATPase and could alter the function of that enzyme. Na,K-ATPases are dimers composed of combinations of four α and three β isoforms. FXYD7 appeared to interact only with α1-β1 isozymes. Studies in injected oocytes showed that unlike other FXYD proteins, such as CHIF (corticosteroid hormone-induced factor), FXYD7 did not alter activation of the Na,K-ATPase by Na + . Rather, FXYD7 decreased the apparent affinity of the Na,K-ATPase for K + . The authors suggest that such modulation of the affinity of the Na,K-ATPase for K + might be important for response to high concentrations of K + that occur after intense neuronal activity. P. Béguin, G. Crambert, F. Monnet-Tschudi, M. Uldry, J.-D. Horisberger, H. Garty, K. Geering, FXYD7 is a brain-specific regulator of Na,K-ATPaseα1-ß isozymes. EMBO J. 21 , 3264-3273 (2002). [Abstract] [Full Text]

CHIF, a member of the FXYD protein family, is a regulator of Na,K-ATPase distinct from the gamma-subunit.

The biological role of small membrane proteins of the new FXYD family is largely unknown. The best characterized FXYD protein is the gamma-subunit of the Na,K-ATPase (NKA) that modulates the Na,K-pump function in the kidney. Here, we report that, similarly to gamma(a) and gamma(b) splice variants, the FXYD protein CHIF (corticosteroid-induced factor) is a type I membrane protein which is associated with NKA in renal tissue, and modulates the Na,K-pump transport when expressed in Xenopus oocytes. In contrast to gamma(a) and gamma(b), which both decrease the apparent Na+ affinity of the Na,K-pump, CHIF significantly increases the Na+ affinity and decreases the apparent K+ affinity due to an increased Na+ competition at external binding sites. The extracytoplasmic FXYD motif is required for stable gamma-subunit and CHIF interaction with NKA, while cytoplasmic, positively charged residues are necessary for the gamma-subunit's association efficiency and for CHIF's functional effects. These data document that CHIF is a new tissue-specific regulator of NKA which probably plays a crucial role in aldosterone-responsive tissues responsible for the maintenance of body Na+ and K+ homeostasis.

Changes of (Na +-K +) dependent ATPase activity in gills and kidneys of two mullets Chelon labrosus (Risso) and Liza ramada (Risso) during fresh water adaptation

Na+/K+–ATPase (NKA) is a primary active pump provides the driving force for ion-transporting systems in the osmoregulatory tissues of teleosts. Therefore, modulation of NKA expression or activity and its regulatory subunit, FXYD protein, is essential for teleosts in salinity adaptation. To understand the mechanisms for modulation of NKA in catadromous fishes, NKA expression and activity, cloning and mRNA expression of FXYD11 (AjFXYD11) were examined in Japanese eel (Anguilla japonica) exposed to fresh water (FW) and seawater (SW; 35‰). Expression and activity of NKA as well as mRNA expression of AjFXYD11 in gills were elevated in SW eel compared to FW eel. Conversely, NKA responses in eel kidneys were higher in FW group than SW group, whereas no significant difference was found in renal AjFXYD11 expression between the two groups. Comparison of NKA activity and AjFXYD11 expression between two osmoregulatory tissues suggested that AjFXYD11 plays a specific, functional role in gills. However, since cortisol plays an important role for regulation of ion transport in teleost SW acclimation and gill AjFXYD11 expression was elevated in SW eel, the organ culture approach was used to study the effect of cortisol on gill AjFXYD11 mRNA expression. Our results revealed that cortisol treatment increased the levels of gill AjFXYD11 transcripts. This finding suggested that cortisol could be involved in the regulation of NKA by altering AjFXYD11 expression during the process of SW acclimation in A. japonica. Taken together, the differential expression of branchial and renal NKA and AjFXYD11 implicated their roles in the osmotic homeostasis of Japanese eel exposed to environments of different salinities.