Biomedical subjects

Elena Arystarkhova

Publications and source records attributed to Elena Arystarkhova.

8 recordsLinked to original sources

PubMed2006

Multiplicity of expression of FXYD proteins in mammalian cells: dynamic exchange of phospholemman and gamma-subunit in response to stress.

Functional properties of Na-K-ATPase can be modified by association with FXYD proteins, expressed in a tissue-specific manner. Here we show that expression of FXYDs in cell lines does not necessarily parallel the expression pattern of FXYDs in the tissue(s) from which the cells originate. While being expressed only in lacis cells in the juxtaglomerular apparatus and in blood vessels in kidney, FXYD1 was abundant in renal cell lines of proximal tubule origin (NRK-52E, LLC-PK1, and OK cells). Authenticity of FXYD1 as a part of Na-K-ATPase in NRK-52E cells was demonstrated by co-purification, co-immunoprecipitation, and co-localization. Induction of FXYD2 by hypertonicity (500 mosmol/kgH(2)O with NaCl for 48 h or adaptation to 700 mosmol/kgH(2)O) correlated with downregulation of FXYD1 at mRNA and protein levels. The response to hypertonicity was influenced by serum factors and entailed, first, dephosphorylation of FXYD1 at Ser(68) (1-5 h) and, second, induction of FXYD2a and a decrease in FXYD1 with longer exposure. FXYD1 was completely replaced with FXYD2a in cells adapted to 700 mosmol/kgH(2)O and showed a significantly decreased sodium affinity. Thus dephosphorylation of FXYD1 followed by exchange of regulatory subunits is utilized to make a smooth transition of properties of Na-K-ATPase. We also observed expression of mRNA for multiple FXYDs in various cell lines. The expression was dynamic and responsive to physiological stimuli. Moreover, we demonstrated expression of FXYD5 protein in HEK-293 and HeLa cells. The data imply that FXYDs are obligatory rather than auxiliary components of Na-K-ATPase, and their interchangeability underlies responses of Na-K-ATPase to cellular stress.

Animals↗

PubMed2005

Na,K-ATPase from mice lacking the gamma subunit (FXYD2) exhibits altered Na+ affinity and decreased thermal stability.

The gamma subunit of the Na,K-ATPase, a 7-kDa single-span membrane protein, is a member of the FXYD gene family. Several FXYD proteins have been shown to bind to Na,K-ATPase and modulate its properties, and each FXYD protein appears to alter enzyme kinetics differently. Different results have sometimes been obtained with different experimental systems, however. To test for effects of gamma in a native tissue environment, mice lacking a functional gamma subunit gene (Fxyd2) were generated. These mice were viable and without observable pathology. Prior work in the mouse embryo showed that gamma is expressed at the blastocyst stage. However, there was no delay in blastocele formation, and the expected Mendelian ratios of offspring were obtained even with Fxyd2-/- dams. In adult Fxyd2-/- mouse kidney, splice variants of gamma that have different nephron segment-specific expression patterns were absent. Purified gamma-deficient renal Na,K-ATPase displayed higher apparent affinity for Na+ without significant change in apparent affinity for K+. Affinity for ATP, which was expected to be decreased, was instead slightly increased. The results suggest that regulation of Na+ sensitivity is a major functional role for this protein, whereas regulation of ATP affinity may be context-specific. Most importantly, this implies that gamma and other FXYD proteins have their effects by local and not global conformation change. Na,K-ATPase lacking the gamma subunit had increased thermal lability. Combined with other evidence that gamma participates in an early step of thermal denaturation, this indicates that FXYD proteins may play an important structural role in the enzyme complex.

Adenosine Triphosphate↗

PubMed2005

Splice variants of the gamma subunit (FXYD2) and their significance in regulation of the Na, K-ATPase in kidney.

The recent discovery of a family of single-span membrane proteins (the FXYD proteins) introduced a new direction to the rather complicated area of regulation of Na, K-ATPase. At least six members of the family have been shown to associate with the Na, K-ATPase in a cell- and tissue-specific manner, while four of them, namely the gamma subunit (FXYD2), CHIF (FXYD4), phospholemman (FXYD1), and dysadherin (FXYD5) have been identified in kidney. All four exhibited different effects on the properties of the pump in heterologous expression systems. Taken along with their non-overlapping expression patterns in the nephron, this provides a potential structural basis for the segment-specific properties of the Na, K-ATPase that had been reported in a number of papers on kidney physiology. This brief review summarizes our own contributions on structure/functional characterization of one of the family members, the gamma subunit (FXYD2). The focus is on splice variants of gamma, their structural similarity and yet distinct effects conferred to Na, K-ATPase.

Alternative Splicing↗

PubMed2004

Stress-induced expression of the gamma subunit (FXYD2) modulates Na,K-ATPase activity and cell growth.

In kidney, the Na,K-ATPase is associated with a single span protein, the gamma 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 gamma by means other than transfection. NRK-52E cells (of proximal tubule origin) do not express gamma as a protein under regular tissue culture conditions. However, when they were exposed to hyperosmotic medium, induction of only the gammaa splice variant was observed, which was accompanied by a reduction in the rate of cell division. Kinetic analysis of stable enzyme properties from control (alpha1beta1) and hypertonicity-treated cultures (alpha1beta1gammaa) 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 alpha1beta1. This effect as well as the reduction in cell growth rate was practically abolished when gamma expression was knocked down using specific small interfering RNA duplexes. Surprisingly, a similar induction of endogenous gammaa 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 gammaa. Taken together, the data imply that induction of gammaa may have adaptive value by being a part of a general cellular response to genotoxic stress.

Adaptation, Physiological↗

PubMed2003

FXYD proteins as regulators of the Na,K-ATPase in the kidney.

The FXYD gene family has seven members in mammals and others in fish. Five of these (FXYD1, FXYD2, FXYD4, FXYD7, and PLMS from shark) have been shown to alter the activity of the Na,K-ATPase, as described by other papers in this volume. The gene structure of FXYD family members suggests assembly from protein domain modules and gene duplication. The gamma subunit is unique in the family for having alternative splice variants in the coding region and can be posttranslationally modified with different final consequences for enzyme properties. The nonoverlapping distribution of gamma and CHIF (FXYD4) in kidney helps to explain physiological differences in Na(+) affinity among nephron segments. We also detected phospholemman (FXYD1) in kidney. By immunofluorescence, it was found in extraglomerular mesangial cells (EM cells) of the juxtaglomerular apparatus and in the afferent arteriole. Contrary to many reports that only alpha1 and beta1 are expressed in the kidney, we found that alpha2 and beta2 are present, although not in any nephron segment. Both were detected in arterioles, and beta2 was found in the EM cells. In contrast, alpha1, beta1, and gamma were found in adjacent macula densa. Phospholemman, alpha2, and beta2 are proposed to have distinct roles in regulating the sodium pump in structures involved in tubuloglomerular feedback.

Animals↗

PubMed2003

Gamma structural variants differentially regulate Na,K-ATPase properties.

Renal Na,K-ATPase is tightly bound to a small regulatory protein, the gamma subunit (FXYD2). In rat, it occurs in two splice variants with different N-termini. Immunolocalization on kidney sections revealed distinct distribution of the gamma splice variants along the rat nephron. Where coexpressed, they coimmunoprecipitated with each other along with the alpha subunit, suggesting assembly in oligomeric complexes. Functional consequences of association with gamma were monitored in stably transfected NRK-52E cells. The outcome was that splice variants can differentially modulate the major intrinsic properties of the Na,K-ATPase under normal and stress-related conditions. The data imply an adaptive physiological mechanism of regulation of renal Na,K-ATPase through modulation of pump properties, gene expression, or both.

Animals↗

PubMed2002

Distribution and oligomeric association of splice forms of Na(+)-K(+)-ATPase regulatory gamma-subunit in rat kidney.

Renal Na(+)-K(+)-ATPase is associated with the gamma-subunit (FXYD2), a single-span membrane protein that modifies ATPase properties. There are two splice variants with different amino termini, gamma(a) and gamma(b). Both were found in the inner stripe of the outer medulla in the thick ascending limb. Coimmunoprecipitation with each other and the alpha-subunit indicated that they were associated in macromolecular complexes. Association was controlled by ligands that affect Na(+)-K(+)-ATPase conformation. In the cortex, the proportion of the gamma(b)-subunit was markedly lower, and the gamma(a)-subunit predominated in isolated proximal tubule cells. By immunofluorescence, the gamma(b)-subunit was detected in the superficial cortex only in the distal convoluted tubule and connecting tubule, which are rich in Na(+)-K(+)-ATPase but comprise a minor fraction of cortex mass. In the outer stripe of the outer medulla and for a short distance in the deep cortex, the thick ascending limb predominantly expressed the gamma(b)-subunit. Because different mechanisms maintain and regulate Na(+) homeostasis in different nephron segments, the splice forms of the gamma-subunit may have evolved to control the renal Na(+) pump through pump properties, gene expression, or both.

Alternative Splicing↗

PubMed2001

Differential regulation of renal Na,K-ATPase by splice variants of the gamma subunit.

Sodium and potassium-exchanging adenosine triphosphatase (Na,K-ATPase) in the kidney is associated with the gamma subunit (gamma, FXYD2), a single-span membrane protein that modulates ATPase properties. Rat and human gamma occur in two splice variants, gamma(a) and gamma(b), with different N termini. Here we investigated their structural heterogeneity and functional effects on Na,K-ATPase properties. Both forms were post-translationally modified during in vitro translation with microsomes, indicating that there are four possible forms of gamma. Site-directed mutagenesis revealed Thr(2) and Ser(5) as potential sites for post-translational modification. Similar modification can occur in cells, with consequences for Na,K-ATPase properties. We showed previously that stable transfection of gamma(a) into NRK-52E cells resulted in reduction of apparent affinities for Na(+) and K(+). Individual clones differed in gamma post-translational modification, however, and the effect on Na(+) affinity was absent in clones with full modification. Here, transfection of gamma(b) also resulted in clones with or without post-translational modification. Both groups showed a reduction in Na(+) affinity, but modification was required for the effect on K(+) affinity. There were minor increases in ATP affinity. The physiological importance of the reduction in Na(+) affinity was shown by the slower growth of gamma(a), gamma(b), and gamma(b') transfectants in culture. The differential influence of the four structural variants of gamma on affinities of the Na,K-ATPase for Na(+) and K(+), together with our previous finding of different distributions of gamma(a) and gamma(b) along the rat nephron, suggests a highly specific mode of regulation of sodium pump properties in kidney.

Adenosine Triphosphatases↗