Identification of Asp804 and Asp808 as Na+ and K+ coordinating residues in α‐subunit of renal Na,K‐ATPase

Pedersen, Per Amstrup; Rasmussen, Jakob H.; Nielsen, Jesper M.; Jørgensen, Peter Leth

doi:10.1016/s0014-5793(96)01381-6

Cited by 56 publications

(52 citation statements)

References 24 publications

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“…Studies on the former enzyme have shown that the carboxyl-selective reagent 4-(diazomethyl)-7-(diethylamino)-coumarin disrupts K ϩ and Na transport (30,31,33,43). Taken together, these findings imply that the M5 glutamyl residue is directly involved in transport by the mammalian Na -ATPase, both of which are required for high affinity cation binding and transport (30,31,33,39,41,43). Unlike its homologues, however, Asp-730 of the H ϩ -ATPase is not detectably involved in transport, since it can be replaced by Ala (as part of an R695A/ D730A double mutant) with little or no effect on ATP hydrolysis or ATP-dependent proton pumping.…”

Section: Mechanism Of Proton Transport: Role Of Charged Residues In Mmentioning

confidence: 97%

Functional Role of Charged Residues in the Transmembrane Segments of the Yeast Plasma Membrane H+-ATPase

Петров¹,

Padmanabha

Nakamoto³

et al. 2000

Journal of Biological Chemistry

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As defined by hydropathy analysis, the membranespanning segments of the yeast plasma membrane H E703Q, E703L, E703D) showed significantly reduced pumping over a wide range of hydrolysis values and thus appeared to be partially uncoupled. At Glu-803, by contrast, one mutant (E803N) was almost completely uncoupled, while another (E803Q) pumped protons at an enhanced rate relative to the rate of ATP hydrolysis. Both Glu-703 and Glu-803 occupy positions at which amino acid substitutions have been shown to affect transport by mammalian P-ATPases. Taken together, the results provide growing evidence that residues in membrane segments 5 and 8 of the P-ATPases contribute to the cation transport pathway and that the fundamental mechanism of transport has been conserved throughout the group.

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Section: Mechanism Of Proton Transport: Role Of Charged Residues In Mmentioning

confidence: 97%

Functional Role of Charged Residues in the Transmembrane Segments of the Yeast Plasma Membrane H+-ATPase

Петров¹,

Padmanabha

Nakamoto³

et al. 2000

Journal of Biological Chemistry

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“…To this end, mutated forms of the enzyme, such as one containing the phosphorylation site mutant, D369N, and cation binding site mutants in the ␣-subunit were expressed, assembled, and assayed using ouabain binding and ATPase activity assays in yeast (97). As a result of these efforts, phosphorylation at D369 was shown to be critical for a major conformational change of the K ϩ -bound enzyme (98,99), and the D804, D808, E327, and E779 residues were found to coordinate sodium and potassium ions as the transporter changes conformation (58,90,100). Multiple other residues were isolated that affect the transporter's ATPase activity (52,109,141).…”

Section: Transporters Namentioning

confidence: 99%

Saccharomyces cerivisiae as a model system for kidney disease: what can yeast tell us about renal function?

Kolb

Buck

Brodsky

2011

American Journal of Physiology-Renal Physiology

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“…The catalytic ␣-subunit of Na,K-ATPase is composed of 10 transmembrane helices (TM). 1 Proteolysis experiments (5, 6) and sitedirected mutagenesis studies indicate that the cation occlusion sites are located within the transmembrane domain, and carboxyl and other oxygen-containing side chain of residues within TM 4, 5, 6, and 8 are crucial for occlusion of cations (7)(8)(9)(10)(11)(12).…”

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confidence: 99%

The Role of Loop 6/7 in Folding and Functional Performance of Na,K-ATPase

Xu¹,

Kane²,

Faller

et al. 2004

Journal of Biological Chemistry

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Alanine substitutions were made for 15 amino acids in the cytoplasmic loop between transmembrane helices 6 and 7 (L6/7) of the human ␣ 1 -subunit of Na,K-ATPase. Most mutations reduced Na,K-ATPase activity by less than 50%; however, the mutations R834A, R837A, and R848A reduced Na,K-ATPase activity by 75, 89, and 66%, respectively. Steady-state phosphoenzyme formation from ATP was reduced in mutants R834A, R837A, and R848A, and R837A also had a faster E 2 P 3 E 2 dephosphorylation rate compared with the wild-type enzyme. Effects of L6/7 mutations on the phosphorylation domain of the protein were also demonstrated by 18 O exchange, which showed that intrinsic rate constants for P i binding and/or reaction with the protein were altered. Although most L6/7 mutations had no effect on the interaction of Na ؉ or K ؉ with Na,K-ATPase, the E825A, E828A, R834A, and R837A mutations reduced the apparent affinity of the enzyme for both Na 3؉ , indicating that Br-TITU 3؉ does not bind to charged residues in L6/7. This observation makes it unlikely that L6/7 functions as a cytoplasmic cation binding site in Na,K-ATPase, and together with the effects of L6/7 mutations on phosphate interactions with the enzyme suggests that L6/7 is important in stabilizing the phosphorylation domain and its relationship to the ion binding sites of the protein.Na,K-ATPase, also called the sodium pump, is a heterodimeric protein consisting of ␣-and ␤-subunits. It pumps 3 Na ϩ out of the cell and 2 K ϩ into the cell during each transport cycle at the expense of ATP hydrolysis, and this activity maintains transmembrane gradients of the ions. Na,K-ATPase belongs to the P-type ATPase family in which the mechanism of action involves formation of a transient, covalently phosphorylated intermediate during the reaction cycle. Other P-type ATPases include sarcoplasmic reticulum and plasma membrane Ca-ATPase, H,K-ATPase found in stomach and colon, and several prokaryotic transport enzymes (1-3). These P-type ATPases have similar structural organization, and amino acid sequences near the sites of ATP binding, phosphorylation, and Mg 2ϩ binding, and residues attributed to cation binding in the transmembrane domain are highly conserved (3, 4). The catalytic ␣-subunit of Na,K-ATPase is composed of 10 transmembrane helices (TM).1 Proteolysis experiments (5, 6) and sitedirected mutagenesis studies indicate that the cation occlusion sites are located within the transmembrane domain, and carboxyl and other oxygen-containing side chain of residues within TM 4, 5, 6, and 8 are crucial for occlusion of cations (7-12).According to the crystal structure of Ca-ATPase (13, 14), the parts of the polypeptide that are exposed in the cytoplasm are organized into three interacting domains designated A, N, and P. The P (phosphorylation) domain is composed of the intracellular loop between TM4 and TM5, with the N-terminal part (roughly Asn 330 -Asn 359 ) connecting to TM4, and the C-terminal part (roughly Lys 605 -Asp 737 ) connecting to TM5. The aspartate residue (Asp 351 ) ...

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Identification of Asp⁸⁰⁴ and Asp⁸⁰⁸ as Na⁺ and K⁺ coordinating residues in α‐subunit of renal Na,K‐ATPase

Cited by 56 publications

References 24 publications

Functional Role of Charged Residues in the Transmembrane Segments of the Yeast Plasma Membrane H+-ATPase

Functional Role of Charged Residues in the Transmembrane Segments of the Yeast Plasma Membrane H+-ATPase

Saccharomyces cerivisiae as a model system for kidney disease: what can yeast tell us about renal function?

The Role of Loop 6/7 in Folding and Functional Performance of Na,K-ATPase

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