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

Kinetics of Transient Pump Currents Generated by the (H,K)-ATPase after an ATP Concentration Jump

(H,K)-ATPase containing membranes from hog stomach were attached to black lipid membranes. Currents induced by an ATP concentration jump were recorded and analyzed. A sum of three exponentials (τ-11 ≈ 400 sec−1, τ -12 ≈ 100 sec−1, τ -13 ≈ 10 sec−1; T = 300 K, pH 6, MgCl2 3 mm, no K+) was fitted to the transient signal. The dependence of the resulting time constants and the peak current on electrolyte composition, ATP conversion rate, temperature, and membrane conductivity was recorded. The results are consistent with a reaction scheme similar to that proposed by Albers and Post for the NaK-ATPase. Based on this model the following assignments were made: τ2 corresponds to ATP binding and exchange with caged ATP. τ 1 describes the phosphorylation reaction E1 · ATP → E1P. The third, slowest time constant τ 3 is tentatively assigned to the E1P → E2P transition. This is the first electrogenic step and is accelerated at high pH and by ATP via a low affinity binding site. The second electrogenic step is the transition from E2K to E1H. The E2K ↔ E1H equilibrium is influenced by potassium with an apparent K 0.5 of 3 mm and by the pH. Low pH and low potassium concentration stabilize the E1 conformation

Why should I trust you? Investigating young children’s spontaneous mistrust in potential deceivers

Children must learn not to trust everyone to avoid being taken advantage of. In the current study, 5- and 7-year-old children were paired with a partner whose incentives were either congruent (cooperative condition) or conflicting (competitive condition) with theirs. Children of both ages were more likely to mistrust information spontaneously provided by the competitive than the cooperative partner, showing a capacity for detecting contextual effects on incentives. However, a high proportion of children, even at age 7, initially trusted the competitive partner. After being misled once, almost all children mistrusted the partner on a second trial irrespective of the partner’s incentives. These results demonstrate that while even school age children are mostly trusting, they are only beginning to spontaneously consider other’s incentives when interpreting the truthfulness of their utterances. However, after receiving false information only once they immediately switch to an untrusting attitude

Cultural variation in young children's social motivation for peer collaboration and its relation to the ontogeny of Theory of Mind.

Children seek and like to engage in collaborative activities with their peers. This social motivation is hypothesized to facilitate their emerging social-cognitive skills and vice versa. Current evidence on the ontogeny of social motivation and its' links to social cognition, however, is subject to a sampling bias toward participants from urban Western populations. Here, we show both cross-cultural variation and homogeneity in three- to eight-year-old children's expressed positive emotions during and explicit preferences for peer collaboration across three diverse populations (urban German, rural Hai||om/Namibia, rural Ovambo/Namibia; n = 240). Children expressed more positive emotions during collaboration as compared to individual activity, but the extent varied across populations. Children's preferences for collaboration differed markedly between populations and across ages: While German children across all ages sought collaboration, Hai||om children preferred to act individually throughout childhood. Ovambo children preferred individual play increasingly with age. Across populations, positive emotions expressed selectively during collaboration, predicted children's social-cognitive skills. These findings provide evidence that culture shapes young children's social motivation for dyadic peer collaboration. At the same time, the positive relation of social motivation and social cognition in early ontogeny appears cross-culturally constant