Rb+ occlusion stabilized by vanadate in gastric H+/K+-ATPase at 25°C

Montes, María Luciana; Spiaggi, Alejandro Javier; Monti, José Luis; Cornelius, Flemming; Olesen, Claus; Garrahan, Patricio J.; Rossi, Rolando

doi:10.1016/j.bbamem.2010.08.022

Cited by 12 publications

(16 citation statements)

References 34 publications

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“…Even when using an SDS-purified G1 fraction or escinpermeabilized preparation (10) (both preparations are completely leaky, as examined by K + -stimulated ATPase activity), incorporation of 86 Rb + into the samples without AlF treatment increased linearly depending on the concentration of 86 RbCl added (SI Appendix, Fig. S4A), which is consistent with the report by Montes et al (11). Therefore, the amount of 86 Rb + binding was calculated as the difference in the amount of incorporated 86 Rb + between AlF-treated and AlF-free samples, to discriminate specific 86 Rb + binding to the AlF-inhibited H + ,K + -ATPase.…”

Section: Methodssupporting

confidence: 89%

“…1B). Our data qualitatively suggest that 86 Rb + binds to the AlF-inhibited H + , K + -ATPase with high affinity (K 0.5 = 16 μM), although the determined stoichiometry is only around 0.15 bound Rb + per ATPase molecule (11), which might be attributable, however, to the exchange of bound 86 Rb + during the washing step of the experiment (see below and SI Appendix, Results and Discussion). The observed high-affinity Rb + binding indicates that the cationbinding site is in an appropriate conformation for Rb + (and also for K + ) coordination, in contrast to the more than fivefold smaller amount of 86 Rb + binding to the E2P ground-state analog, the beryllium fluoride (BeF)-inhibited enzyme (Fig.…”

Section: Resultsmentioning

confidence: 58%

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Cryo-EM structure of gastric H ⁺ ,K ⁺ -ATPase with a single occupied cation-binding site

Abe

Tani²,

Friedrich

et al. 2012

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

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Gastric H + ,K + -ATPase is responsible for gastric acid secretion. ATPdriven H + uptake into the stomach is efficiently accomplished by the exchange of an equal amount of K + , resulting in a luminal pH close to 1. Because of the limited free energy available for ATP hydrolysis, the stoichiometry of transported cations is thought to vary from 2H + /2K + to 1H + /1K + per hydrolysis of one ATP molecule as the luminal pH decreases, although direct evidence for this hypothesis has remained elusive. Here, we show, using the phosphate analog aluminum fluoride (AlF) and a K + congener (Rb + ), the 8-Å resolution structure of H + ,K + -ATPase in the transition state of dephosphorylation, (Rb + )E2∼AlF, which is distinct from the preceding Rb + -free E2P state. A strong density located in the transmembrane cation-binding site of (Rb + )E2∼AlF highly likely represents a single bound Rb + ion, which is clearly different from the Rb + -free E2AlF or K + -bound (K + )E2∼AlF structures. Measurement of radioactive 86 Rb + binding suggests that the binding stoichiometry varies depending on the pH, and approximately half of the amount of Rb + is bound under acidic crystallization conditions compared with at a neutral pH. These data represent structural and biochemical evidence for the 1H + /1K + /1ATP transport mode of H + ,K + -ATPase, which is a prerequisite for generation of the 10 6 -fold proton gradient in terms of thermodynamics. Together with the released E2P-stabilizing interaction between the β subunit's N terminus and the P domain observed in the (Rb + )E2∼AlF structure, we propose a refined vectorial transport model of H + ,K + -ATPase, which must prevail against the highly acidic state of the gastric lumen.electron crystallography | P-type ATPases | membrane proteins | bioenergetics L ike other P-type ATPases (1), the vectorial cation transport of gastric H + ,K + -ATPase (2) is accomplished by cyclical conformational changes of the enzyme (abbreviated as E), generally described using an E1/E2 nomenclature based on the PostAlbers scheme for Na + ,K + -ATPase (3) (SI Appendix, Fig. S1). In contrast to the closely related Na + ,K + -ATPase, which exchanges three Na + for two K + ions in an electrogenic transport reaction, gastric H + ,K + -ATPase operates electroneutrally, although its transport stoichiometry (2H + /2K + /ATP or 1H + /1K + /ATP) has remained controversial (4, 5). A measurement of the proton transport (5) revealed an H + /ATP ratio of 2 at neutral pH. Accordingly, two K + ions must be counter transported during a single turnover of the transport cycle, accompanied by the hydrolysis of one ATP molecule (2H + /2K + /ATP). The reported free energy for ATP hydrolysis of the gastric secretory membrane [−13 kcal/mol (4)] provides sufficient energy to achieve a maximum change in pH (ΔpH) of 4.7 units when two H + ions are transported per hydrolysis of one ATP molecule; thus, the generation of pH 1 (ΔpH > 6 units in the stomach) with a 2H + /2K + / ATP stoichiometry is thermodynamically impossible. Therefore,...

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Section: Methodssupporting

confidence: 89%

Section: Resultsmentioning

confidence: 58%

Cryo-EM structure of gastric H ⁺ ,K ⁺ -ATPase with a single occupied cation-binding site

Abe

Tani²,

Friedrich

et al. 2012

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

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“…We tested this by treating the microsomal membranes with alamethicin, a peptide that forms large pores allowing the passage of organic molecules of considerable size such as ATP. Alamethicin has been used previously in transport studies of the SERCA (7) and determinations of occlusion of Rb ϩ in the H ϩ /K ϩ -ATPase (19). Following incubation of microsomal membranes with different concentrations of alamethicin for 30 min at 25°C, retained calcium was measured as a function of time from 1.5 to 8.5 s (Fig.…”

Section: Calcium Retained By Microsomal Vesicles Expressing Pmca-mentioning

confidence: 99%

Calcium Occlusion in Plasma Membrane Ca2+-ATPase

Ferreira-Gomes

González-Lebrero

Fuente

et al. 2011

Journal of Biological Chemistry

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In this work, we set out to identify and characterize the calcium occluded intermediate(s) of the plasma membrane Ca 2؉ -ATPase (PMCA) to study the mechanism of calcium transport. To this end, we developed a procedure for measuring the occlusion of Ca 2؉ in microsomes containing PMCA. This involves a system for overexpression of the PMCA and the use of a rapid mixing device combined with a filtration chamber, allowing the isolation of the enzyme and quantification of retained calcium. Measurements of retained calcium as a function of the Ca 2؉ concentration in steady state showed a hyperbolic dependence with an apparent dissociation constant of 12 ؎ 2.2 M, which agrees with the value found through measurements of PMCA activity in the absence of calmodulin. When enzyme phosphorylation and the retained calcium were studied as a function of time in the presence of La III (inducing accumulation of phosphoenzyme in the E 1 P state), we obtained apparent rate constants not significantly different from each other. Quantification of EP and retained calcium in steady state yield a stoichiometry of one mole of occluded calcium per mole of phosphoenzyme. These results demonstrate for the first time that one calcium ion becomes occluded in the E 1 P-phosphorylated intermediate of the PMCA.The plasma membrane calcium ATPase (PMCA) 3 is a calmodulin-modulated P-type ATPase responsible for the maintenance of low intracellular concentrations of Ca 2ϩ in most eukaryotic cells. It couples the transport of Ca 2ϩ out of cells with the hydrolysis of ATP into ADP and inorganic phosphate. PMCAs consist of a single polypeptide chain of 127,000 to 137,000 Da. Mammalian PMCAs are encoded by four separate genes (PMCA1-4), and additional isoforms are generated via alternative RNA splicing, which augments the number of variants to Ͼ20 (1).The current kinetic model for PMCA function proposes that the enzyme exists in two main conformations, E 1 and E 2 . E 1 has a high affinity for Ca 2ϩ and is readily phosphorylated by ATP, whereas E 2 has a low affinity for Ca 2ϩ and can be phosphorylated by P i . After binding of intracellular Ca 2ϩ to high affinity sites, E 1 can be phosphorylated by ATP with formation of the intermediate E 1 P. After a conformational transition to E 2 P, Ca 2ϩ would be released to the extracellular medium from low affinity sites, followed by the hydrolysis of the phosphoenzyme to E 2 and a new conformational transition to E 1 (Fig. 1) (2). During some stages of the reaction cycle, Ca 2ϩ becomes occluded, i.e. trapped in the enzyme machinery while it is transported from one side to the other side of the membrane. The principal aim of this study is to identify and kinetically characterize the intermediate(s) of the PMCA containing occluded calcium. Evidence for occlusion in Na,K-ATPase and sarcoplasmic reticulum Ca 2ϩ -ATPase (SERCA) has been well established (3), and a good deal of information exists about the occlusion and deocclusion steps of the transported cations in these pumps. Na ϩ and K ϩ are occluded in the E ...

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“…Interestingly, decavanadate [V 10 O 28 ] 6− is a more potent Ca 2+ ATPase inhibitor than monomeric vanadate [207][208][209]. The oxidation of a cysteine residue through reduction of the vanadate apparently is the inhibition mechanism of decavanadate to Ca 2+ -ATPase [205][206][207][208][209]. Myosin is considered as an ATPase because contains a motor domain comprising two binding sites responsible for interacting with the actin and ATP hydrolysis (the head); meanwhile, the intermediate domain arm increases the conformational change caused by ATP hydrolysis and is responsible for the binding of regulatory light chains like calmodulin.…”

Section: Vanadium and Intracellular Proteins Interactionmentioning

confidence: 99%

“…Monomeric vanadate mimics the transition state for the phosphate hydrolysis [213], blocking myosin by the ADP-phosphate intermediate state. Decavanadate also inhibits myosin ATPase and Ca 2+ -ATPase [205][206][207][208]. Decavanadate induces the formation of the intermediate myosin-MgATP-V10 complex blocking the contractile cycle, most probably in the pre-hydrolysis state [211].…”

Section: Vanadium and Intracellular Proteins Interactionmentioning

confidence: 99%

Vanadium in Biological Action: Chemical, Pharmacological Aspects, and Metabolic Implications in Diabetes Mellitus

Treviño

Díaz

Sánchez-Lara

et al. 2018

Biol Trace Elem Res

241

126

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Vanadium compounds have been primarily investigated as potential therapeutic agents for the treatment of various major health issues, including cancer, atherosclerosis, and diabetes. The translation of vanadium-based compounds into clinical trials and ultimately into disease treatments remains hampered by the absence of a basic pharmacological and metabolic comprehension of such compounds. In this review, we examine the development of vanadium-containing compounds in biological systems regarding the role of the physiological environment, dosage, intracellular interactions, metabolic transformations, modulation of signaling pathways, toxicology, and transport and tissue distribution as well as therapeutic implications. From our point of view, the toxicological and pharmacological aspects in animal models and humans are not understood completely, and thus, we introduced them in a physiological environment and dosage context. Different transport proteins in blood plasma and mechanistic transport determinants are discussed. Furthermore, an overview of different vanadium species and the role of physiological factors (i.e., pH, redox conditions, concentration, and so on) are considered. Mechanistic specifications about different signaling pathways are discussed, particularly the phosphatases and kinases that are modulated dynamically by vanadium compounds because until now, the focus only has been on protein tyrosine phosphatase 1B as a vanadium target. Particular emphasis is laid on the therapeutic ability of vanadium-based compounds and their role for the treatment of diabetes mellitus, specifically on that of vanadate-and polioxovanadate-containing compounds. We aim at shedding light on the prevailing gaps between primary scientific data and information from animal models and human studies.

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Rb+ occlusion stabilized by vanadate in gastric H+/K+-ATPase at 25°C

Cited by 12 publications

References 34 publications

Cryo-EM structure of gastric H ⁺ ,K ⁺ -ATPase with a single occupied cation-binding site

Cryo-EM structure of gastric H ⁺ ,K ⁺ -ATPase with a single occupied cation-binding site

Calcium Occlusion in Plasma Membrane Ca2+-ATPase

Vanadium in Biological Action: Chemical, Pharmacological Aspects, and Metabolic Implications in Diabetes Mellitus

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Rb+ occlusion stabilized by vanadate in gastric H+/K+-ATPase at 25°C

Cited by 12 publications

References 34 publications

Cryo-EM structure of gastric H + ,K + -ATPase with a single occupied cation-binding site

Cryo-EM structure of gastric H + ,K + -ATPase with a single occupied cation-binding site

Calcium Occlusion in Plasma Membrane Ca2+-ATPase

Vanadium in Biological Action: Chemical, Pharmacological Aspects, and Metabolic Implications in Diabetes Mellitus

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Cryo-EM structure of gastric H ⁺ ,K ⁺ -ATPase with a single occupied cation-binding site

Cryo-EM structure of gastric H ⁺ ,K ⁺ -ATPase with a single occupied cation-binding site