Role of Fxyd Proteins in Ion Transport

Garty, Haim; Karlish, Steven J. D.

doi:10.1146/annurev.physiol.68.040104.131852

Cited by 224 publications

(218 citation statements)

References 120 publications

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“…Therefore, if one were to attribute the biphasic behavior to enzyme heterogeneity caused by the presence of FXYD proteins, this would be in contradiction to previous observations (35,36). Furthermore, in their recent review of FXYD protein interaction with the Na + ,K + -ATPase, Garty and Karlish (34) stated that the effect of FXYD proteins on the kinetic parameters of the enzyme are modest, usually only about 2-fold. In contrast, the reciprocal relaxation times that we have observed for the two phases in the absence of ATP ( Figure 1A) are a factor of over 20-fold different (i.e., 10.4 s -1 for the fast phase and 0.49 s -1 for the slow phase), an order of magnitude greater than FXYD protein effects.…”

Section: Resultsmentioning

confidence: 88%

Allosteric Effect of ATP on Na⁺,K⁺-ATPase Conformational Kinetics

2007

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The kinetics of the E 2 f E 1 conformational change of unphosphorylated Na + ,K + -ATPase was investigated via the stopped-flow technique using the fluorescent label RH421 (pH 7.4, 24°C). The enzyme was pre-equilibrated in a solution containing 25 mM histidine and 0.1 mM EDTA to stabilize the E 2 conformation. When rabbit enzyme was mixed with 130 mM NaCl alone or with 130 mM NaCl and varying concentrations of Na 2 ATP simultaneously, a fluorescence decrease was observed. In the absence of ATP, the fluorescence decrease followed a biexponential time course, but at ATP concentrations after mixing of g50 µM, the fluorescence transient could be adequately fitted by a single exponential. On the basis of the agreement between theoretical simulations and experimental traces, we propose that in the absence of bound ATP the conformational transition occurs as a two step reversible process within a protein dimer, E 2 :E 2 f E 2 :E 1 f E 1 :E 1 . In the presence of 130 mM NaCl, the sum of the forward and backward rate constants for the E 2 :E 2 f E 2 :E 1 and E 2 :E 1 f E 1 :E 1 transitions were found to be 10.4 ((1.0) and 0.49 ((0.02) s -1 , respectively. At saturating concentrations of ATP, however, the transition occurs in a single reversible step with the sum of its forward and backward rate constants equal to 35.2 ((0.3) s -1 . It was found that ATP acting at a high affinity site (K d ≈ 0.25 µM), stimulated the reverse reaction, E 1 ATP f E 2 ATP, in addition to its known allosteric low affinity (K d ≈ 71 µM) stimulation of the forward reaction, E 2 ATP f E 1 ATP. Na + ,K + -ATPase 1 was the first ion pump to be discovered (1), and it is one of the most fundamentally important enzymes of animal physiology. The electrochemical potential gradient for Na + ions, which the enzyme maintains, is used as the driving force for numerous secondary transport systems. Examples include the Na + channels in the nerve, which allow the action potential to be produced, the Na + -glucose cotransporter, which is responsible for glucose uptake in intestinal cells, and the Na + /Ca 2+ exchanger in the heart, which plays an important role in muscle relaxation. Na + ,K + -ATPase, furthermore, contributes to the osmotic regulation of cell volume and is a major determinant of body temperature. For all of these functions, the enzyme derives its energy from the hydrolysis of ATP, which is coupled to Na + transport.For many years, it has been known that ATP also has an allosteric effect on the enzyme. Under physiological conditions, ATP binds to the E 2 (K + ) 2 conformation of the enzyme and accelerates the release of K + ions to the cytoplasm without undergoing any hydrolysis. This was first demonstrated by Karlish and Yates (2), who measured the rate of the E 2 f E 1 conformational transition, which occurs simultaneously with K + release, via the stopped-flow technique, utilizing the change in the enzyme's tryptophan fluorescence as the means of detection. This stimulation of the E 2 f E 1 transition by ATP has since been confirmed...

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Section: Resultsmentioning

confidence: 88%

Allosteric Effect of ATP on Na⁺,K⁺-ATPase Conformational Kinetics

2007

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“…The β-subunit is a glycosylated polypeptide that promotes folding and positioning of the protein into the basolateral plasma membrane. A third subunit termed γ, also known as FXYD, is not necessary for the catalytic function of NKA, but apparently acts to adapt the kinetic properties of sodium and potassium transport for the functions of different cell types (Garty and Karlish, 2006). Vertebrates express at least four isoforms of the α-subunit that vary in their kinetic properties for Na + , K + and ATP binding, sensitivity to ouabain and calcium, and their regulation by intracellular second messengers (Blanco and Mercer, 1998).…”

Section: Introductionmentioning

confidence: 99%

Differential regulation of sodium–potassium pump isoforms during smolt development and seawater exposure of Atlantic salmon

McCormick

Regish²,

Christensen

et al. 2013

Journal of Experimental Biology

100

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SUMMARYFreshwater and seawater isoforms of the alpha subunit of Na + /K + -ATPase (NKA) have previously been identified in gill ionocytes of Atlantic salmon (Salmo salar). In the present study we examine the abundance and cellular localization of these isoforms during the parr-smolt transformation, a developmental process that is preparatory for seawater entry. The abundance of NKAα1a was lower in smolts than in parr, remained relatively constant during spring and decreased in summer. NKAα1b increased tenfold in smolts during spring, peaking in late April, coincident with downstream migration and increased salinity tolerance. NKAα1b increased a further twofold after seawater exposure of smolts, whereas NKAα1a decreased by 98%. The abundance of NKAα1b-positive, and NKAα1b and NKAα1a co-labeled ionocytes increased during smolt development, whereas the number of NKAα1a cells decreased. After seawater exposure of smolts, NKAα1b-positive ionocytes increased, NKAα1a-positive cells decreased, and co-labeled cells disappeared. Plasma growth hormone and cortisol increased during spring in smolts, but not in parr, peaking just prior to the highest levels of NKAα1b. The results indicate that the increase in the abundance of NKAα1b during smolt development is directly linked to the increase in salinity tolerance that occurs at this stage, but that significant changes also occur after seawater exposure. Spring increases in circulating levels of growth hormone and cortisol indicate that these hormones may be instrumental in upregulating NKAα1b during smolt development.

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“…In this paper, the methods are described and illustrated with examples from the FXYD proteins, a family of tissue-specific and physiological-state-specific subunits of the Na,KATPase, that help to regulate the distribution of Na and K ion concentrations across animal cell membranes [17][18][19]. The FXYD protein sequences are highly conserved through evolution, and are characterized by a 35-amino acid FXYD homology domain, which includes the short signature motif PFXYD (Pro, Phe, X, Tyr, Asp) and the transmembrane domain (Fig.…”

Section: Introductionmentioning

confidence: 99%

NMR of membrane proteins in micelles and bilayers: The FXYD family proteins

Franzin

Gong

Thai

et al. 2007

Methods

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Determining the atomic resolution structures of membrane proteins is of particular interest in contemporary structural biology. Helical membrane proteins constitute one-third of the expressed proteins encoded in a genome, many drugs have membrane-bound proteins as their receptors, and mutations in membrane proteins result in human diseases. Although integral membrane proteins provide daunting technical challenges for all methods of protein structure determination, nuclear magnetic resonance (NMR) spectroscopy can be an extremely versatile and powerful method for determining their structures and characterizing their dynamics, in lipid environments that closely mimic the cell membranes. Once milligram amounts of isotopically labeled protein are expressed and purified, micelle samples can be prepared for solution NMR analysis, and lipid bilayer samples can be prepared for solid-state NMR analysis. The two approaches are complementary and can provide detailed structural and dynamic information. This paper describes the steps for membrane protein structure determination using solution and solid-state NMR. The methods for protein expression and purification, sample preparation and NMR experiments are described and illustrated with examples from the FXYD proteins, a family of regulatory subunits of the Na, KATPase.

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Role of Fxyd Proteins in Ion Transport

Cited by 224 publications

References 120 publications

Allosteric Effect of ATP on Na⁺,K⁺-ATPase Conformational Kinetics

Allosteric Effect of ATP on Na⁺,K⁺-ATPase Conformational Kinetics

Differential regulation of sodium–potassium pump isoforms during smolt development and seawater exposure of Atlantic salmon

NMR of membrane proteins in micelles and bilayers: The FXYD family proteins

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Role of Fxyd Proteins in Ion Transport

Cited by 224 publications

References 120 publications

Allosteric Effect of ATP on Na+,K+-ATPase Conformational Kinetics

Allosteric Effect of ATP on Na+,K+-ATPase Conformational Kinetics

Differential regulation of sodium–potassium pump isoforms during smolt development and seawater exposure of Atlantic salmon

NMR of membrane proteins in micelles and bilayers: The FXYD family proteins

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Allosteric Effect of ATP on Na⁺,K⁺-ATPase Conformational Kinetics

Allosteric Effect of ATP on Na⁺,K⁺-ATPase Conformational Kinetics