Electrogenic Properties of the Endogenous and of Modified Torpedo Na+/K+-Pumps in Xenopus Oocytes: The Access Channel for External Cations

Schwarz, Wolfgang; Vasilets, Larisa A.; Omay, Halit; Efthymiadis, Athina; Rettinger, Jürgen; Elsner, Silke

doi:10.1007/978-3-642-72511-1_88

Cited by 4 publications

(3 citation statements)

References 23 publications

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“…Hao et al (11) observed no increase in Ca 2ϩ flux when the E m was collapsed in reconstituted proteoliposomes. These results are similar to those obtained on the Na ϩ pump in the absence of extracellular Na ϩ and the presence of saturating K ϩ (27,29). (The comparable conditions for the Ca 2ϩ pump are in the absence of extracellular Ca 2ϩ and saturating extracellular H ϩ .)…”

Section: Discussionsupporting

confidence: 87%

“…There is strong electrophysiological evidence for an access well for the Na ϩ pump (3,9,12,23,26,27,29). The Na ϩ pump and the Ca 2ϩ pump are in the same family.…”

Section: Discussionmentioning

confidence: 99%

“…The Na ϩ pump is in the same family as the PMCA and, in contrast to the Ca 2ϩ pump, the potential dependence has been studied extensively (3,9,12,23,27,29). In one version of the high-field access channel model, the first Na ϩ to exit the pump moves through a high-field access channel (23,29). Most of the potential drop across the protein (the transmembrane potential) occurs across this high-field access channel in this conformation.…”

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confidence: 99%

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Probing the extracellular release site of the plasma membrane calcium pump

Wilson

Huang

et al. 2000

American Journal of Physiology-Cell Physiology

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The plasma membrane Ca(2+) pump is known to mediate Ca(2+)/H(+) exchange. Extracellular protons activated (45)Ca(2+) efflux from human red blood cells with a half-maximal inhibition constant of 2 nM when the intracellular pH was fixed. An increase in pH from 7.2 to 8.2 decreased the IC(50) for extracellular Ca(2+) from approximately 33 to approximately 6 mM. Changing the membrane potential by >54 mV had no effect on the IC(50) for extracellular Ca(2+). This argues against Ca(2+) release through a high-field access channel. Extracellular Ni(2+) inhibited Ca(2+) efflux with an IC(50) of 11 mM. Extracellular Cd(2+) inhibited with an IC(50) of 1. 5 mM, >10 times better than Ca(2+). The Cd(2+) IC(50) also decreased when the pH was raised from 7.1 to 8.2, consistent with Ca(2+), Cd(2+), and H(+) competing for the same site. The higher affinity for inhibition by Ni(2+) and Cd(2+) is consistent with a histidine or cysteine as part of the release site. The cysteine reagent 2-(trimethylammonium)ethyl methanethiosulfonate did not inhibit Ca(2+) efflux. Our results are consistent with the notion that the release site contains a histidine.

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Section: Discussionsupporting

confidence: 87%

“…There is strong electrophysiological evidence for an access well for the Na ϩ pump (3,9,12,23,26,27,29). The Na ϩ pump and the Ca 2ϩ pump are in the same family.…”

Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

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Probing the extracellular release site of the plasma membrane calcium pump

Wilson

Huang

et al. 2000

American Journal of Physiology-Cell Physiology

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Membrane Transport in Single Cells

Jennings

Milanick

1997

Comprehensive Physiology

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The sections in this article are: Examples of Preparations of Single Cells Large Cells that Can Be Studied Individually Blood Cells Cells Prepared by Dissociating Intact Tissue Permanent Cell Lines and Expression Systems Methods for Measuring Transport Tracer Influx and Efflux: Definition of Flux Definition of Permeability Coefficient Unstirred Layers Multiple Intracellular Compartments Optical Methods for Measuring Transport Ion‐Sensitive Microelectrodes Membrane Potential Nuclear Magnetic Resonance Transport Mechanisms Rationale for Investigating Transport Mechanisms Catalytic Cycles Anion Exchanger Red Blood Cell Anion Transport Electrically Silent Obligatory Exchange Kinetic Mechanism of Anion Exchange Intrinsic Asymmetries Electrical Properties of the Exchange Pathway Sodium Pump Electrically Silent and Electrogenic Na Pump Modes Pump Stoichiometry Kinetic Mechanism of the Na Pump Electrical Properties of the Na Pump Summary of Transport Mechanisms Integrative Transport Studies in Single Cells: Cell‐Volume Regulation Pump‐Leak Paradigm Volume‐Sensitive Transport Processes Detection of Cell Volume Transduction of Volume Signal to Transport Proteins

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Significance of the glutamic acid residues Glu334, Glu959, and Glu960 of the α subunits of Torpedo Na+,K+ pumps for transport activity and ouabain binding

Vasilets

Takeda

Kawamura

et al. 1998

Biochimica et Biophysica Acta (BBA) - Biomembranes

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Glutamic acid residues in transmembrane segments of the alpha subunit of the Na+,K+-ATPase have been discussed as possible candidates for the binding sites of the transported cations. Here we report on effects of mutations of Glu334, Glu959, and Glu960 to alanine in ouabain-sensitive (OS) as well as ouabain-resistant (OR) ATPases of Torpedo electroplax expressed in Xenopus oocytes. All mutants are incorporated to about the same extend as the wild-type ATPases into the plasma membrane. None of the mutations produces complete inhibition of transport activity as judged from measurements of 86Rb+ uptake, membrane current, and ATPase activity. After conversion of OS to OR by mutation of the bordering residues of the first extracellular loop Gln118 to Arg and Asp129 to Asn, the Km value for inhibition by ouabain increases to 59 microM. Substitution of Glu334 to Ala in the OR pump variant restores ouabain sensitivity with a Km value of 0.12 microM, which is similar to that of the endogenous Xenopus pump. After substitution of Glu960 by Ala in the OR pump, ouabain sensitivity is partially restored. The Km values for pump stimulation by external K+ appear to be reduced in the OR compared to the OS pump. Mutation of Glu959 and Glu960 to Ala has no pronounced effects on the potential-dependent Km values at external pH 7.8; only in the Glu959-mutated OR pump, the apparent Km at 0 mV is raised. We conclude that none of the mutated glutamic acid residues is essential for cation coordination, but that GIu334, and in part also Glu960, seems to be involved in preserving the ouabain-resistant conformation of the enzyme.

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Electrogenic Properties of the Endogenous and of Modified Torpedo Na+/K+-Pumps in Xenopus Oocytes: The Access Channel for External Cations

Cited by 4 publications

References 23 publications

Probing the extracellular release site of the plasma membrane calcium pump

Probing the extracellular release site of the plasma membrane calcium pump

Membrane Transport in Single Cells

Significance of the glutamic acid residues Glu334, Glu959, and Glu960 of the α subunits of Torpedo Na+,K+ pumps for transport activity and ouabain binding

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