A nervous system-specific isotype of the beta subunit of Na+,K(+)-ATPase expressed during early development of Xenopus laevis.

Good, Peter J.; Richter, Klaus; Dawid, Igor B.

doi:10.1073/pnas.87.23.9088

Cited by 103 publications

(49 citation statements)

References 47 publications

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“…In outside-out patches excised from uninjected Xenopus oocytes, the presence of 100 M ouabain effectively prevents current signals from endogenous, ouabain-sensitive, Xenopus Na͞K pumps (believed to comprise Xenopus ␣1 and ␤3 subunits (19,20) in response to application of picomolar concentrations of PTX (see Fig. 8, which is published as supporting information on the PNAS web site).…”

Section: Resultsmentioning

confidence: 99%

Ouabain affinity determining residues lie close to the Na/K pump ion pathway

Artigas

Gadsby

2006

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

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The Na͞K pump establishes essential ion concentration gradients across animal cell membranes. Cardiotonic steroids, such as ouabain, are specific inhibitors of the Na͞K pump. We exploited the marine toxin, palytoxin, to probe both the ion translocation pathway through the Na͞K pump and the site of its interaction with ouabain. Palytoxin uncouples the pump's gates, which normally open strictly alternately, thus allowing both gates to sometimes be open, so transforming the pump into an ion channel. Palytoxin therefore permits electrophysiological analysis of even a single Na͞K pump. We used outside-out patch recording of Xenopus ␣1␤3 Na͞K pumps, which were made ouabain-resistant by point mutation, after expressing them in Xenopus oocytes. Endogenous, ouabain-sensitive, Xenopus ␣1␤3 Na͞K pumps were silenced by continuous exposure to ouabain. We found that side-chain charge of two residues at either end of the ␣ subunit's first extracellular loop, known to make a major contribution to ouabain affinity, strongly influenced conductance of single palytoxin-bound pumpchannels by an electrostatic mechanism. The effects were mimicked by modification of cysteines introduced at those two positions with variously charged methanethiosulfonate reagents. The consequences of these modifications demonstrate that both residues lie in a wide vestibule near the mouth of the pump's ion pathway. Bound ouabain protects the site with the strongest influence on conductance from methanethiosulfonate modification, while leaving the site with the weaker influence unprotected. The results suggest a method for mapping the footprint of bound cardiotonic steroid on the extracellular surface of the Na͞K pump.Na,K-ATPase ͉ palytoxin ͉ single-channel conductance ͉ cysteine modification ͉ electrostatic mechanism

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Section: Resultsmentioning

confidence: 99%

Ouabain affinity determining residues lie close to the Na/K pump ion pathway

Artigas

Gadsby

2006

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

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“…At least two types of isoforms, (31 and (32, have been characterized in rats by cDNA cloning ( 11), and recently the existence of the (3 isoform has been reported ( 12). The function of the subunit remains unknown, but it has been suggested that the subunit plays a role in the integration of the a subunits into the membrane (6).…”

Section: Discussionmentioning

confidence: 99%

Regulation of Na,K-adenosine triphosphatase gene expression by sodium ions in cultured neonatal rat cardiocytes.

Yamamoto¹,

Ikeda²,

Seino³

et al. 1993

J. Clin. Invest.

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Na,K-ATPase (NaK-pump) plays an important role in the regulation of intracellular ion composition. The purpose of this study is to determine whether Na' regulates the levels of mRNA coding for NaK-ATPase a and fi subunits in cultured neonatal rat cardiocytes. We measured intracellular Na+ levels (QNa'Ij) in cardiocytes using a Na'-sensitive fluorescence dye (SBFI). 1 mM ouabain caused a significant increase in [Nalji in cardiocytes; from 12.8±03 to 28.8±1.8 mM. Exposure of cardiocytes to 1 mM ouabain resulted in a three-to fourfold increase in al, a2, and a3 mRNA accumulation, and an approximate two-fold increase in fB1 mRNA accumulation. A maximum elevation was reached at 60 min in both cases. The ouabain-induced al mRNA accumulation was still observed in the Ca2"-free culture medium. Exposure of cardiocytes to 10 gM monensin in the absence of extracellular Ca2" also resulted in a threefold increase in al mRNA accumulation. The increased al mRNA expression by 1 mM ouabain was associated with a fourfold increase in al subunit protein accumulation. Transfection experiments with chimeric plasmids containing 5'-flanking sequences of al, a2, and a3 isoform genes and a luciferase reporter gene revealed that 1 mM ouabain caused a twofold increase in luciferase activity in each a system. These results suggest that Na+ directly regulates NaK-ATPase gene expression in cardiocytes. The transfection study further supports the premise that Na+-responsive elements are located within the 5'-flanking sequences of each a isoform gene. (J.

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“…The near full-length a, cDNA (-45 to +3349 bp = &wt) from Xcnopus laevis previously cloned from an A6 kidney cell library [9] and a /3x cDNA cloned from a Xenopus neurula library [7,8] were inserted into the pSDS vector [6]. We have previously shown that both the PI September 1991 Fig.…”

Section: Deletion Mutnnl At the N Terminus Of The ~1 Isofarmmentioning

confidence: 99%

Functional expression of N‐terminal truncated α‐subunits of Na,K‐ATPase in Xenopus laevis oocytes

et al. 1991

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N‐terminal deletion mutants of Na,K‐ATPase α1 isoforms initiating translation at Met34 (α1T1) or at Met43 (α1T2) were expressed in X. laevis oocytes. Compared to β3 cRNA injected controls, the co‐expression of α1wt, α1T1, α1T2 with β3 subunits results in a 2‐ to 3‐fold increase of ouabain binding sites, parallelled by a concomitant increase in Na,K‐pump current. The apparent K½ for potassium activation of the α1T2/β3 Na,K‐pumps is significantly higher than that of the α1wt/β3 or α1T1/β3 Na,K‐pumps expressed at the cell surface. Total deletion of the lysine‐rich N‐terminal domain thus allows the expression of active Na,K‐pump but with distinct cation transport properties.

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A nervous system-specific isotype of the beta subunit of Na+,K(+)-ATPase expressed during early development of Xenopus laevis.

Cited by 103 publications

References 47 publications

Ouabain affinity determining residues lie close to the Na/K pump ion pathway

Ouabain affinity determining residues lie close to the Na/K pump ion pathway

Regulation of Na,K-adenosine triphosphatase gene expression by sodium ions in cultured neonatal rat cardiocytes.

Functional expression of N‐terminal truncated α‐subunits of Na,K‐ATPase in Xenopus laevis oocytes

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