Control of Gastric H,K-ATPase Activity by Cations, Voltage and Intracellular pH Analyzed by Voltage Clamp Fluorometry in Xenopus Oocytes

Whereas electrogenic partial reactions of the Na,K-ATPase have been studied in depth, much less is known about the influence of the membrane potential on the electroneutrally operating gastric H,K-ATPase. In this work, we investigated site-specifically fluorescence-labeled H,K-ATPase expressed in Xenopus oocytes by voltage clamp fluorometry to monitor the voltage-dependent distribution between E1P and E2P states and measured Rb+ uptake under various ionic and pH conditions. The steady-state E1P/E2P distribution, as indicated by the voltage-dependent fluorescence amplitudes and the Rb+ uptake activity were highly sensitive to small changes in intracellular pH, whereas even large extracellular pH changes affected neither the E1P/E2P distribution nor transport activity. Notably, intracellular acidification by approximately 0.5 pH units shifted V0.5, the voltage, at which the E1P/E2P ratio is 50∶50, by −100 mV. This was paralleled by an approximately two-fold acceleration of the forward rate constant of the E1P→E2P transition and a similar increase in the rate of steady-state cation transport. The temperature dependence of Rb+ uptake yielded an activation energy of ∼90 kJ/mol, suggesting that ion transport is rate-limited by a major conformational transition. The pronounced sensitivity towards intracellular pH suggests that proton uptake from the cytoplasmic side controls the level of phosphoenzyme entering the E1P→E2P conformational transition, thus limiting ion transport of the gastric H,K-ATPase. These findings highlight the significance of cellular mechanisms contributing to increased proton availability in the cytoplasm of gastric parietal cells. Furthermore, we show that extracellular Na+ profoundly alters the voltage-dependent E1P/E2P distribution indicating that Na+ ions can act as surrogates for protons regarding the E2P→E1P transition. The complexity of the intra- and extracellular cation effects can be rationalized by a kinetic model suggesting that cations reach the binding sites through a rather high-field intra- and a rather low-field extracellular access channel, with fractional electrical distances of ∼0.5 and ∼0.2, respectively

ALTARUPPSATSEN I HOLMS KYRKA PÅ DAL - EN STILANALYS

Holms kyrka på Dal är en medeltida sockenkyrka. Dekoration av kyrkorummet utfördes under 1700-talet av provinsiella träsnidare och målare i en stil som kommit att kallas bondbarock. Altaruppsatsen skulpterad i trä omger altartavlan som är uppdelad i två fält, varav det undre är en kalvariegrupp och den övre visar Jesus i Getsemane. Överstelöjtnant Gustav von Mentzer uppges ha donerat altaruppsatsen till kyrkan år 1727 men det saknas uppgifter om vem bildhuggaren var. Hans George Schüffner har stofferat den i trä skulpterade predikstolen från år 1742 tillskriven Isac Schiulström. Vid en jämförande stilanalys av altaruppsatsen i Holms kyrka och åtta andra altaruppsatser av Nils Falk och Isac Schiulström framkommer att altaruppsatsen i Holms kyrkan med stor sannolikhet är av bildhuggaren Nils Falk

Large scale expression, purification and 2D crystallization of recombinant plant plasma membrane H⁺-ATPase

P-type ATPases convert chemical energy into electrochemical gradients that are used to energize secondary active transport. Analysis of the structure and function of P-type ATPases has been limited by the lack of active recombinant ATPases in quantities suitable for crystallographic studies aiming at solving their three-dimensional structure. We have expressed Arabidopsis thaliana plasma membrane H+-ATPase isoform AHA2, equipped with a His6-tag, in the yeast Saccharomyces cerevisiae. The H+-ATPase could be purified both in the presence and in the absence of regulatory 14-3-3 protein depending on the presence of the diterpene fusicoccin which specifically induces formation of the H+-ATPase/14-3-3 protein complex. Amino acid analysis of the purified complex suggested a stoichiometry of two 14-3-3 proteins per H+-ATPase polypeptide. The purified H+-ATPase readily formed two-dimensional crystals following reconstitution into lipid vesicles. Electron cryo-microscopy of the crystals yielded a projection map at ∼8 Å resolution, the p22121 symmetry of which suggests a dimeric protein complex. Three distinct regions of density of approximately equal size are apparent and may reflect different domains in individual molecules of AHA2