Extracellular Domains, Transmembrane Segments, and Intracellular Domains Interact To Determine the Cation Selectivity of Na,K- and Gastric H,K-ATPase

Mense, Martin; Rajendran, V.; Blostein, Rhoda; Caplan, Michael J.

doi:10.1021/bi025819z

Cited by 15 publications

(12 citation statements)

References 33 publications

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“…Similar to our results, Mense et al (37) found this substitution to be responsible for conferring the Na,K-ATPase with a Na-independent ATPase activity and a dependency toward protons. In that work, however, Thr345 alone was important but not sufficient to induce the enzymatic changes mentioned and substitutions in the Na,K-ATPase of two additional residues (Leu319 and Thr340), as well as the TM3-TM4 ectodomain of the H,K-ATPase were also required (38). Our results show that substitutions confined to transmembrane domain 4 are able to alter Na + selectivity of the enzyme.…”

Section: Discussionmentioning

confidence: 50%

“…The function of the TMAll mutant indicates that transmembrane domains are not sufficient to confer Na + selectivity to the Na,K-ATPase and other domains are involved. Our observation confirms previous results by Mense at al., who show that cytoplasmic, as well as extracellular, domains interact to create the particular properties of P-type ATPases (38).…”

Section: Discussionmentioning

confidence: 99%

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Residues within Transmembrane Domains 4 and 6 of the Na,K-ATPase α Subunit Are Important for Na⁺ Selectivity

Sánchez

Blanco

2004

Biochemistry

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The Na,K- and H,K-ATPases are plasma membrane enzymes responsible for the active exchange of extracellular K(+) for cytoplasmic Na(+) or H(+), respectively. At present, the structural determinants for the specific function of these ATPases remain poorly understood. To investigate the cation selectivity of these ATPases, we constructed a series of Na,K-ATPase mutants in which residues in the membrane spanning segments of the alpha subunit were changed to the corresponding residues common to gastric H,K-ATPases. Thus, mutants were created with substitutions in transmembrane domains TM1, TM4, TM5, TM6, TM7, and TM8 independently or together (designated TMAll). The function of each mutant was assessed after coexpression with the beta subunit in Sf-9 cells using baculoviruses. The enzymatic properties of TM1, TM7, and TM8 mutants were similar to the wild-type Na,K-ATPase, and while TM5 showed modest changes in apparent affinity for Na(+), TM4, TM6, and TMAll displayed an abnormal activity. This resulted in a Na(+)-independent hydrolysis of ATP, a 2-fold higher K(0.5) for Na(+) activation, and the ability to function at low pH. These results suggest a loss of discrimination for Na(+) over H(+) for the enzymes. In addition, TM4, TM6, and TMAll mutants exhibited a 1.5-fold lower affinity for K(+) and a 4-5-fold decreased sensitivity to vanadate. Altogether, these results provide evidence that residues in transmembrane domains 4 and 6 of the alpha subunit of the Na,K-ATPase play an important role in determining the specific cation selectivity of the enzyme and also its E1/E2 conformational equilibrium.

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Section: Discussionmentioning

confidence: 50%

Section: Discussionmentioning

confidence: 99%

Residues within Transmembrane Domains 4 and 6 of the Na,K-ATPase α Subunit Are Important for Na⁺ Selectivity

Sánchez

Blanco

2004

Biochemistry

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“…3. This part includes only animal IIC ATPases that, generally, may be considered bona fide a-HKAs and a-NKAs (Mense et al 2002;Jorgensen et al 2003). There is no bootstrap support for the separation of both sub-trees (i.e., below 50%).…”

Section: Resultsmentioning

confidence: 99%

Evolutionary history of Na,K-ATPases and their osmoregulatory role

2009

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The Na/K pump, or Na,K-ATPase, is a key enzyme to the homeostasis of osmotic pressure, cell volume, and the maintenance of electrochemical gradients. Its alpha subunit, which holds most of its functions, belongs to a large family of ATPases known as P-type, and to the subfamily IIC, which also includes H,K-ATPases. In this study, we attempt to describe the evolutionary history of IIC ATPases by doing phylogenetic analysis with most of the currently available protein sequences (over 200), and pay special attention to the relationship between their diversity and their osmoregulatory role. We include proteins derived from many completed or ongoing genome projects, many of whose IIC ATPases have not been phylogenetically analyzed previously. We show that the most likely origin of IIC proteins is prokaryotic, and that many of them are present in non-metazoans, such as algae, protozoans or fungi. We also suggest that the pre-metazoan ancestor, represented by the choanoflagellate Monosiga brevicollis, whose genome has been sequenced, presented at least two IIC-type proteins. One of these proteins would have given rise to most current animal IIC ATPases, whereas the other apparently evolved into a lineage that, so far, has only been found in nematodes. We also propose that early deuterostomes presented a single IIC gene, from which all the extant diversity of vertebrate IIC proteins originated by gene and genome duplications.

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“…1). Identification of the subunit domains involved in assembly and trafficking of Na,KATPase has been approached by immune precipitation experiments with truncated ␤-subunits (12,13) and chimeras between the Na,K-ATPase and gastric H,K-ATPase ␣-subunits (14,15).…”

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confidence: 99%

Interactions between Na,K-ATPase α-Subunit ATP-binding Domains

Costa¹,

Gatto²,

Kaplan³

2003

Journal of Biological Chemistry

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The reaction mechanism of the Na,K-ATPase is thought to involve a number of ligand-induced conformational changes. The specific amino acid residues responsible for binding many of the important ligands have been identified; however, details of the specific conformational changes produced by ligand binding are largely undescribed. The experiments described in this paper begin to identify interactions between domains of the Na,K-ATPase ␣-subunit that depend on the presence of particular ligands. The major cytoplasmic loop (between TM4 and TM5), which we have previously shown contains the ATP-binding domain, was overexpressed in bacteria either with a His 6 tag or as a fusion protein with glutathione S-transferase. We have observed that these polypeptides associate in the presence of MgATP. Incubation with [␥-32 P]ATP under conditions that result in phosphorylation of the full-length Na,K-ATPase did not result in 32 P incorporation into either the His 6 tag or glutathione S-transferase fusion proteins. The MgATPinduced association was strongly inhibited by prior modification of the fusion proteins with fluorescein isothiocyanate or by simultaneous incubation with 10 M eosin, indicating that the effect of MgATP is due to interactions within the nucleotide-binding domain. These data are consistent with Na,K-ATPase associating within cells via interactions in the nucleotide-binding domains. Although any functional significance of these associations for ion transport remains unresolved, they may play a role in cell function and in modulating interactions between the Na,K-ATPase and other proteins.

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Extracellular Domains, Transmembrane Segments, and Intracellular Domains Interact To Determine the Cation Selectivity of Na,K- and Gastric H,K-ATPase

Cited by 15 publications

References 33 publications

Residues within Transmembrane Domains 4 and 6 of the Na,K-ATPase α Subunit Are Important for Na⁺ Selectivity

Residues within Transmembrane Domains 4 and 6 of the Na,K-ATPase α Subunit Are Important for Na⁺ Selectivity

Evolutionary history of Na,K-ATPases and their osmoregulatory role

Interactions between Na,K-ATPase α-Subunit ATP-binding Domains

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Extracellular Domains, Transmembrane Segments, and Intracellular Domains Interact To Determine the Cation Selectivity of Na,K- and Gastric H,K-ATPase

Cited by 15 publications

References 33 publications

Residues within Transmembrane Domains 4 and 6 of the Na,K-ATPase α Subunit Are Important for Na+ Selectivity

Residues within Transmembrane Domains 4 and 6 of the Na,K-ATPase α Subunit Are Important for Na+ Selectivity

Evolutionary history of Na,K-ATPases and their osmoregulatory role

Interactions between Na,K-ATPase α-Subunit ATP-binding Domains

Contact Info

Product

Resources

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Residues within Transmembrane Domains 4 and 6 of the Na,K-ATPase α Subunit Are Important for Na⁺ Selectivity

Residues within Transmembrane Domains 4 and 6 of the Na,K-ATPase α Subunit Are Important for Na⁺ Selectivity