Mechanistic Basis for Kinetic Differences between the Rat α1, α2, and α3 Isoforms of the Na,K-ATPase

Segall, Laura; Daly, Stewart E.; Blostein, Rhoda

doi:10.1074/jbc.m103720200

Cited by 53 publications

(36 citation statements)

References 39 publications

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…6) but with little variation seen among the combinations ␣3␤1, ␣3␤2, and ␣3␤3. These various parameters were evaluated in Xenopus oocytes where all of the different combinations of human Na pump isoforms were expressed (28); similar type studies have also been carried out by others using different expression systems with either human (29) or rat (30,31) Na pump isoforms where the differences in the kinetic parameters of ␣1 compared with ␣3 are similar to those given in the Inset of Fig. 6.…”

Section: Discussionmentioning

confidence: 99%

Na pump isoforms in human erythroid progenitor cells and mature erythrocytes

Hoffman

Wickrema

Potapova

et al. 2002

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

View full text Add to dashboard Cite

This study is aimed at identifying the Na pump isoform composition of human erythroid precursor cells and mature human erythrocytes. We used purified and synchronously growing human erythroid progenitor cells cultured for 7-14 days. RNA was extracted from the progenitor cells on different days and analyzed by RT-PCR. The results showed that only the ␣1, ␣3, ␤2, and ␤3 subunit isoforms and the ␥ modulator were present. Northern analysis of the erythroid progenitor cells again showed that ␤2 but not ␤1 or ␣2 isoforms were present. The erythroid cells display a unique ␤ subunit expression profile (called ␤-profiling) in that they contain the message for the ␤2 isoform but not ␤1, whereas leukocytes and platelets are known to have the message for the ␤1 but not for the ␤2 isoform. This finding is taken to indicate that our preparations are essentially purely erythroid and free from white cell contamination. Western analysis of these cultured progenitor cells confirmed the presence of ␣1, ␣3, (no ␣2), ␤2, ␤3, and ␥ together now with clear evidence that ␤1 protein was also present at all stages. Western analysis of the Na pump from mature human erythrocyte ghosts, purified by ouabain column chromatography, has also shown that ␣1, ␣3, ␤1, ␤2, ␤3, and ␥ are present. Thus, the Na pump isoform composition of human erythroid precursor cells and mature erythrocytes contains the ␣1 and ␣3 isoforms of the ␣ subunit, the ␤1, ␤2, and ␤3 isoforms of the ␤ subunit, and the ␥ modulator.

show abstract

Section: Discussionmentioning

confidence: 99%

Na pump isoforms in human erythroid progenitor cells and mature erythrocytes

Hoffman

Wickrema

Potapova

et al. 2002

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

View full text Add to dashboard Cite

show abstract

“…Catalytic turnover was estimated from the ratio of V max to maximal phosphoenzyme (EP max ) as in ref. 17, except that 50 mM NaCl (or KCl for baseline EP) was included during the room temperature incubation with oligomycin. Transport assays were carried out on cells grown in 24-well plates as described in ref.…”

Section: Methodsmentioning

confidence: 99%

“…In terms of activity, the contribution of ␣2 is even lower (Ϸ10%), given the Ϸ50% lower catalytic turnover of ␣2 compared with ␣1 (17,31). In fact, in the heterozygote ␣2 knockout mouse, bulk intracellular Na ϩ in the astrocyte is not altered (32).…”

Section: How Do the Functional Changes Explain The Fhm2 Disease Phenomentioning

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.

Self Cite

View full text Add to dashboard Cite

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 ...

show abstract

“…Catalytic turnover was estimated from the ratio of V max to EP max as in Ref. 11. Unless indicated otherwise, transport assays were carried out as described by Munzer et al (19).…”

Section: Methodsmentioning

confidence: 99%

Kinetic Alterations due to a Missense Mutation in the Na,K-ATPase α2 Subunit Cause Familial Hemiplegic Migraine Type 2

Segall

Scanzano

Kaunisto

et al. 2004

Journal of Biological Chemistry

Self Cite

View full text Add to dashboard Cite

A number of missense mutations in the ATP1A2 gene, which encodes the Na,K-ATPase ␣2 subunit, have been identified in familial hemiplegic migraine with aura. Loss of function and haploinsufficiency have been the suggested mechanisms in mutants for which functional analysis has been reported. This paper describes a kinetic analysis of mutant T345A, recently identified in a detailed genetic analysis of a large Finnish family (Kaunisto, M. A., Harno, H., Vanmolkot, K. R., Gargus, J. J., Sun, G., Hamalainen, E., Liukkonen, E., Kallela, M., van den Maagdenberg, A. M., Frants, R. R., Farkkila, M., Palotie, A., and Wessman, M. (2004) Neurogenetics 5, 141-146). Introducing T345A into the conserved rat ␣2 enzyme does not alter cell growth or catalytic turnover but causes a substantial decrease in apparent K ؉ affinity (2-fold increase in K 0.5(K ؉ ) ). In view of the location of Thr-345 in the cytoplasmic stalk domain adjacent to transmembrane segment 4, the 2-fold increase in K 0.5(K ؉ ) is probably due to T345A replacement altering K ؉ occlusion/deocclusion. Faster K ؉ deocclusion of the mutant via the E 2 (K) ؉ ATP 3 E 1 ⅐ATP ؉ K ؉ partial reaction is evidenced in (i) a marked increase (300%) in K ؉ stimulation of Na-ATPase at micromolar ATP, (ii) a 4-fold decrease in K ATP , and (iii) only a modest increase (ϳ3-fold) in I 50 for vanadate, which was used as a probe of the steady state E 1 /E 2 conformational equilibrium. We suggest that the decreased apparent K ؉ affinity is the basis for a reduced rate of extracellular K ؉ removal, which delays the recovery phase of nerve impulse transmission in the central nervous system and, thereby, the clinical picture of migraine with aura. This is the first demonstration of a mutation that leads to a disease associated with a kinetically altered but fully functional Na,K-ATPase, refining the molecular mechanism of pathogenesis in familial hemiplegic migraine.Familial hemiplegic migraine (FHM) 1 is a rare autosomal dominant form of migraine with aura. This disorder is usually associated with hemiparesis and can be accompanied with clinical features ranging from ataxia to epileptic seizures. This genetically heterogeneous disease has been traced to at least two loci, FHM1 and FHM2. FHM1, which accounts for over 50% of all FHM families, has been traced to chromosome 19p13 and associated with missense mutations in the CACNA1A gene encoding the ␣1 subunit of the voltage-dependent neuronal (P/Q type) calcium channel. A recent breakthrough in migraine genetics is the discovery of missense mutations in the ATP1A2 gene on chromosome 1q23 that encodes the ␣2 isoform of the Na,K-ATPase. This finding gives strong support to the notion that FHM is caused by disruption of normal cation transport. The Na,K-ATPase is an integral membrane protein complex that comprises a large catalytic ␣ subunit of ϳ110 kDa as well as a smaller, highly glycosylated ␤ subunit that ensures the proper folding and mooring of ␣ in the plasma membrane. This P-type ion pump catalyzes the ATP-driven exchange of intrace...

show abstract

Mechanistic Basis for Kinetic Differences between the Rat α1, α2, and α3 Isoforms of the Na,K-ATPase

Cited by 53 publications

References 39 publications

Na pump isoforms in human erythroid progenitor cells and mature erythrocytes

Na pump isoforms in human erythroid progenitor cells and mature erythrocytes

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

Kinetic Alterations due to a Missense Mutation in the Na,K-ATPase α2 Subunit Cause Familial Hemiplegic Migraine Type 2

Contact Info

Product

Resources

About