Structure/Function Analysis of the Amino-terminal Region of the α1 and α2 Subunits of Na,K-ATPase

Daly, Stewart E.; Lane, Lois K.; Blostein, Rhoda

doi:10.1074/jbc.271.39.23683

Cited by 58 publications

(107 citation statements)

References 26 publications

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“…Thus, at micromolar ATP concentrations sufficient to saturate only the high affinity phosphorylation site the response of Na-ATPase to K ϩ is a sensitive means to characterize mutantspecific differences in the K ϩ -deocclusion pathway of the reaction cycle [E 2 (K ϩ ) 33 E 1 ϩ K ϩ ] that becomes rate-limiting under these conditions (2,17). As shown previously and summarized in Table I (upper panel), a low concentration of K ϩ (1 mM KCl) inhibits the Na-ATPase activity of ␣1 but stimulates that of ␣1M32 and ␣1E233K with a further and notably synergistic stimulation of the double mutant ␣1M32E233K.…”

Section: Resultsmentioning

confidence: 99%

“…It should be noted that our original intent was to investigate 5 individual divergent regions contained within residues 429 -565 (see "Experimental Procedures," cassettes I-V) on the E 1 /E 2 equilibrium of ␣1. Because the individual domains showed no effect on the K ϩ inhibition of Na-ATPase of ␣1 relevant to the K ϩ deocclusion pathway, 2 we constructed an ␣1/␣2 chimera where all 5 cassettes of ␣2* were substituted into ␣1. Two additional chimeras were constructed.…”

Section: Resultsmentioning

confidence: 99%

“…HeLa cells expressing the mutant ␣ enzymes were amplified in Dulbecco's modified Eagle's medium plus 10% newborn calf serum, 100 units/mg penicillin G, 100 g/ml streptomycin, and 1 M ouabain as described previously (2).…”

Section: Methodsmentioning

confidence: 99%

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Structural Basis for α1 Versus α2 Isoform-distinct Behavior of the Na,K-ATPase

Segall¹,

Javaid²,

Carl³

et al. 2003

Journal of Biological Chemistry

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We showed earlier that the kinetic behavior of the ␣2 isoform of the Na,K-ATPase differs from the ubiquitous ␣1 isoform primarily by a shift in the steady-state E 1 /E 2 equilibrium of ␣2 in favor of E 1 form(s). The aim of the present study was to identify regions of the ␣ chain that confer the ␣1/␣2 distinct behavior using a mutagenesis and chimera approach. Criteria to assess shifts in conformational equilibrium included (i) K ؉ sensitivity of Na-ATPase measured at micromolar ATP, under which condition E 2 (K ؉ ) 3 E 1 ؉ K ؉ becomes rate-limiting, (ii) changes in K ATP for low affinity ATP binding, (iii) vanadate sensitivity of Na,K-ATPase activity, and (iv) the rate of the partial reaction E 1 P 3 E 2 P. We first confirmed that interactions between the cytoplasmic domains of ␣2 that modulate conformational shifts are fundamentally similar to those of ␣1, suggesting that the predilection of ␣2 for E 1 state(s) is due to differences in primary structure of the two isoforms. Kinetic behavior of the ␣1/␣2 chimeras indicates that the difference in E 1 /E 2 poise of the two isoforms cannot be accounted for by their notably distinct N termini, but rather by the front segment extending from the cytoplasmic N terminus to the C-terminal end of the extracellular loop between transmembranes 3 and 4, with a lesser contribution of the ␣1/␣2 divergent portion within the M4-M5 loop near the ATP binding domain. In addition, we show that the E 1 shift of ␣2 results primarily from differences in the conformational transition of the dephosphoenzyme, (E 2 (K ؉ ) 3 E 1 ؉ K ؉ ), rather than phosphoenzyme (E 1 P 3 E 2 P).The Na,K-ATPase or sodium pump is an integral membrane protein complex found in the plasma membrane of virtually all animal cells. It catalyzes the exchange of three intracellular Na ϩ ions for two extracellular K ϩ ions using the energy of hydrolysis of one molecule of ATP. Consequently, the sodium pump plays an essential role in the maintenance of the electrochemical alkali cation gradients, providing the driving force for the transport of various nutrients into the cell. The Na,KATPase is a member of the family of P-type ATPases, which during the course of their catalytic cycle undergo phosphorylation and dephosphorylation of a conserved aspartate residue located in the large catalytic loop between transmembrane segments 4 and 5 of the catalytic ␣ subunit. During the catalytic cycle both dephospho-and phosphoenzymes undergo conformational transitions commonly referred to as E 1 7 E 2 and E 1 P 7 E 2 P, respectively. In addition to the large catalytic ␣ subunit, the Na,K-ATPase comprises a smaller, highly glycosylated ␤ subunit that is important for the proper folding of ␣ and its insertion into the plasma membrane. At present, four isoforms of ␣ and three isoforms of ␤ have been described, and these are distributed in a tissue-and developmentally dependent manner.The ␣2 isoform is located primarily in skeletal muscle and in brain, predominantly in glial cells. Our earlier studies indicated that it differs from t...

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

See 1 more Smart Citation

Structural Basis for α1 Versus α2 Isoform-distinct Behavior of the Na,K-ATPase

Segall¹,

Javaid²,

Carl³

et al. 2003

Journal of Biological Chemistry

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“…Thus, the ratio of V max ͞EP max in WT is 4,619 Ϯ 426 min Ϫ1 , similar to that reported in ref. 21 and is reduced to 2,042 Ϯ 374 and 880 Ϯ 247 min Ϫ1 for R689Q and M731T replacements, respectively.…”

Section: Resultsmentioning

confidence: 99%

Alterations in the α2 isoform of Na,K-ATPase associated with familial hemiplegic migraine type 2

Segall

Mezzetti

Scanzano

et al. 2005

Proc. Natl. Acad. Sci. U.S.A.

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A number of missense mutations in the Na,K-ATPase ␣2 catalytic subunit have been identified in familial hemiplegic migraine with aura. Two alleles (L764P and W887R) showed loss-of-function, whereas a third (T345A) is fully functional but with altered Na,KATPase kinetics. This study describes two additional mutants, R689Q and M731T, originally identified by Vanmolkot et al. , which we show here to also be functional and kinetically altered. Both mutants have reduced catalytic turnover and increased apparent affinity for extracellular K ؉ . For both R689Q and M731T, sensitivity to vanadate inhibition is decreased, suggesting that the steadystate E 1 7 E2 poise of the enzyme is shifted toward E1. Whereas the K ATP is not affected by the R689Q replacement, the M731T mutant has an increase in apparent affinity for ATP. Analysis of the structural changes effected by T345A, R689Q, and M731T mutations, based on homologous replacements in the known crystal structure of the sarcoplasmic reticulum Ca-ATPase, provides insights into the molecular bases for the kinetic alterations. It is suggested that the disease phenotype is the consequence of lowered molecular activity of the ␣2 pump isoform due to either decreased K ؉ affinity (T345A) or catalytic turnover (R689Q and M731T), thus causing a delay in extracellular K ؉ clearance and͞or altered localized Ca 2؉ handling͞signaling secondary to reduced activity in colocalized Na ؉ ͞Ca 2؉ exchange.sodium pump kinetics ͉ missense mutations ͉ ATP1A2 gene T he Na,K-ATPase catalyzes the ATP-driven exchange of three intracellular Na ϩ ions for two extracellular K ϩ ions across the plasma membrane of virtually all animal cells and is essential to the maintenance of the electrochemical alkali cation gradients that are dissipated by ion channels in the propagation of action potentials (for recent reviews, see refs. 1 and 2). The catalytic cycle of this P type ion pump involves phosphorylation and dephosphorylation of a conserved aspartate residue in its catalytic ␣ subunit and conformational transitions of phosphoand dephosphoenzyme, commonly referred to as E 1 P 7 E 2 P and E 1 7 E 2 conformational transitions, respectively (see Scheme 1). Four isoforms of ␣ and three isoforms of ␤ have been described thus far. All are distributed in a tissue-and developmentally dependent manner. In adult mammals, ␣2 is located principally in skeletal muscle and brain, in particular in glial cells, and, to a lesser extent, in heart, adipocytes, and the eye (see refs. 3-6).The discovery of the association of missense mutations in the ATP1A2 gene on chromosome 1q23 that encodes the Na,KATPase ␣2 isoform with familial hemiplegic migraine (FHM) has been an important breakthrough in that it reflects a disease caused by a kinetically altered sodium pump and is therefore an important lead in migraine pathophysiology. Although a migraine is a common polygenic disorder, FHM is a rare autosomal dominant form of migraine with aura and is usually additionally associated with hemiparesis and other clinical features ...

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“…2 and 3 show that the mutations induced larger affinity decreases in the low affinity effect than in the high affinity effect (see also Table II, a). Given these findings, it would be of interest to investigate whether the mutation affects the sensitivity of Na/K-ATPase to vanadate, an analogue of P i that binds to the E 2 form of Na/K-ATPase (35,36). Fig.…”

Section: Effect Of Substitution On the Apparent Low Affinity Atp Effementioning

confidence: 99%

The Amino Acid Sequence 442GDASE446 in Na/K-ATPase Is an Important Motif in Forming the High and Low Affinity ATP Binding Pockets

Imagawa

Kaya

Taniguchi

2003

Journal of Biological Chemistry

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.5 ) and the apparent K m for Mg 2؉ , Na ؉ , K ؉ , and vanadate in Na/K-ATPase were also estimated. For all the mutants, the value for ATP was ϳ2-10-fold larger than that of the wild type. While the turnover number for Na/K-ATPase activity were unaffected or reduced by 20ϳ50% in mutants G442(A/P) and D443A. Although both affinities for ATP effects were reduced as a result of the mutations, the ratio, K m l /K m h , for each mutant was 1.3ϳ3.7, indicating that these mutations had a greater impact on the low affinity ATP effect than on the high affinity effect. Each K m P value with the turnover number suggests that these mutations favor the binding of pNPP over that of ATP. These data and others indicate that the sequence 442 GDASE 446 in the ATP binding pocket is an important motif that it is involved in both the high and low affinity ATP effects rather than in free Mg 2؉ , Na ؉ , and K ؉ effects.

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Structure/Function Analysis of the Amino-terminal Region of the α1 and α2 Subunits of Na,K-ATPase

Cited by 58 publications

References 26 publications

Structural Basis for α1 Versus α2 Isoform-distinct Behavior of the Na,K-ATPase

Structural Basis for α1 Versus α2 Isoform-distinct Behavior of the Na,K-ATPase

Alterations in the α2 isoform of Na,K-ATPase associated with familial hemiplegic migraine type 2

The Amino Acid Sequence 442GDASE446 in Na/K-ATPase Is an Important Motif in Forming the High and Low Affinity ATP Binding Pockets

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