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

PLoS ONE ◽  
2012 ◽  
Vol 7 (3) ◽  
pp. e33645 ◽  
Author(s):  
Katharina L. Dürr ◽  
Neslihan N. Tavraz ◽  
Thomas Friedrich
Keyword(s):  
Atpase Activity ◽  
Voltage Clamp ◽  
Intracellular Ph ◽  
Xenopus Oocytes

scholarly journals Na+/H+ and Na+/NH4+ exchange activities of zebrafish NHE3b expressed in Xenopus oocytes

2014 ◽  
Vol 306 (5) ◽  
pp. R315-R327 ◽  
Author(s):  
Yusuke Ito ◽  
Akira Kato ◽  
Taku Hirata ◽  
Shigehisa Hirose ◽  
Michael F. Romero
Keyword(s):  
Voltage Clamp ◽  
Fresh Water ◽  
Intracellular Ph ◽  
Concentration Gradient ◽  
Xenopus Oocytes ◽  
Apical Membrane ◽  
Free Media ◽  
Exchange Activity

Zebrafish Na+/H+ exchanger 3b (zNHE3b) is highly expressed in the apical membrane of ionocytes where Na+ is absorbed from ion-poor fresh water against a concentration gradient. Much in vivo data indicated that zNHE3b is involved in Na+ absorption but not leakage. However, zNHE3b-mediated Na+ absorption has not been thermodynamically explained, and zNHE3b activity has not been measured. To address this issue, we overexpressed zNHE3b in Xenopus oocytes and characterized its activity by electrophysiology. Exposure of zNHE3b oocytes to Na+-free media resulted in significant decrease in intracellular pH (pHi) and intracellular Na+ activity ( aNai). aNai increased significantly when the cytoplasm was acidified by media containing CO2-HCO3− or butyrate. Activity of zNHE3b was inhibited by amiloride or 5-ethylisopropyl amiloride (EIPA). Although the activity was accompanied by a large hyperpolarization of ∼50 mV, voltage-clamp experiments showed that Na+/H+ exchange activity of zNHE3b is electroneutral. Exposure of zNHE3b oocytes to medium containing NH3/NH4+ resulted in significant decreases in pHi and aNai and significant increase in intracellular NH4+ activity, indicating that zNHE3b mediates the Na+/NH4+ exchange. In low-Na+ (0.5 mM) media, zNHE3b oocytes maintained aNai of 1.3 mM, and Na+-influx was observed when pHi was decreased by media containing CO2-HCO3− or butyrate. These results provide thermodynamic evidence that zNHE3b mediates Na+ absorption from ion-poor fresh water by its Na+/H+ and Na+/NH4+ exchange activities.

scholarly journals Extracellular Hco3− Dependence of Electrogenic Na/Hco3 Cotransporters Cloned from Salamander and Rat Kidney

2000 ◽  
Vol 115 (5) ◽  
pp. 533-546 ◽  
Author(s):  
Irina I. Grichtchenko ◽  
Michael F. Romero ◽  
Walter F. Boron
Keyword(s):  
Membrane Potential ◽  
Voltage Clamp ◽  
Xenopus Oocytes ◽  
Rat Kidney ◽  
Outward Current ◽  
Membrane Vesicles ◽  
Disulfonic Acid ◽  
Ambystoma Tigrinum ◽  
Ph Titration ◽  
Electrode Voltage

We studied the extracellular [HCOabstract 3 −] dependence of two renal clones of the electrogenic Na/HCO3 cotransporter (NBC) heterologously expressed in Xenopus oocytes. We used microelectrodes to measure the change in membrane potential (ΔVm) elicited by the NBC cloned from the kidney of the salamander Ambystoma tigrinum (akNBC) and by the NBC cloned from the kidney of rat (rkNBC). We used a two-electrode voltage clamp to measure the change in current (ΔI) elicited by rkNBC. Briefly exposing an NBC-expressing oocyte to HCOabstract 3 −/CO2 (0.33–99 mM HCOabstract 3−, pHo 7.5) elicited an immediate, DIDS (4,4-diisothiocyanatostilbene-2,2-disulfonic acid)-sensitive and Na+-dependent hyperpolarization (or outward current). In ΔVm experiments, the apparent Km for HCOabstract 3− of akNBC (10.6 mM) and rkNBC (10.8 mM) were similar. However, under voltage-clamp conditions, the apparent Km for HCOabstract 3− of rkNBC was less (6.5 mM). Because it has been reported that SOabstract 3=/HSO abstract 3− stimulates Na/HCO3 cotransport in renal membrane vesicles (a result that supports the existence of a COabstract 3= binding site with which SOabstract 3= interacts), we examined the effect of SOabstract 3=/HSO abstract 3− on rkNBC. In voltage-clamp studies, we found that neither 33 mM SOabstract 4= nor 33 mM SOabstract 3 =/HSOabstract 3− substantially affects the apparent Km for HCO abstract 3−. We also used microelectrodes to monitor intracellular pH (pHi) while exposing rkNBC-expressing oocytes to 3.3 mM HCOabstract 3 −/0.5% CO2. We found that SO abstract 3=/HSOabstract 3 − did not significantly affect the DIDS-sensitive component of the pHi recovery from the initial CO2 -induced acidification. We also monitored the rkNBC current while simultaneously varying [CO2]o, pHo, and [COabstract 3=]o at a fixed [HCOabstract 3−]o of 33 mM. A Michaelis-Menten equation poorly fitted the data expressed as current versus [COabstract 3=]o . However, a pH titration curve nicely fitted the data expressed as current versus pHo. Thus, rkNBC expressed in Xenopus oocytes does not appear to interact with SOabstract 3 =, HSOabstract 3−, or COabstract 3=.

Effect of PCMBS on CO2permeability of Xenopus oocytes expressing aquaporin 1 or its C189S mutant

1998 ◽  
Vol 275 (6) ◽  
pp. C1481-C1486 ◽  
Author(s):  
Gordon J. Cooper ◽  
Walter F. Boron
Keyword(s):  
Carbonic Anhydrase ◽  
Intracellular Ph ◽  
Xenopus Oocytes ◽  
Membrane Lipid ◽  
Wild Type ◽  
Aquaporin 1 ◽  
Gas Channel ◽  
A Cell ◽  
Pass Through ◽  
Rule Out

A recent study on Xenopus oocytes [N. L. Nakhoul, M. F. Romero, B. A. Davis, and W. F. Boron. Am. J. Physiol. 274 ( Cell Physiol. 43): C543–548, 1998] injected with carbonic anhydrase showed that expressing aquaporin 1 (AQP1) increases by ∼40% the rate at which exposing the cell to CO2 causes intracellular pH to fall. This observation is consistent with several interpretations. Overexpressing AQP1 might increase apparent CO2 permeability by 1) allowing CO2 to pass through AQP1, 2) stimulating injected carbonic anhydrase, 3) enhancing the CO2 solubility of the membrane’s lipid, or 4) increasing the expression of a native “gas channel.” The purpose of the present study was to distinguish among these possibilities. We found that expressing the H2O channel AQP1 in Xenopus oocytes increases the CO2 permeability of oocytes in an expression-dependent fashion, whereas expressing the K+ channel ROMK1 has no effect. The mercury derivative p-chloromercuriphenylsulfonic acid (PCMBS), which inhibits the H2O movement through AQP1, also blocks the AQP1-dependent increase in CO2 permeability. The mercury-insensitive C189S mutant of AQP1 increases the CO2 permeability of the oocyte to the same extent as does the wild-type channel. However, the C189S-dependent increase in CO2permeability is unaffected by treatment with PCMBS. These data rule out options 2–4 listed above. Thus our results suggest that CO2passes through the pore of AQP1 and are the first data to demonstrate that a gas can enter a cell by a means other than diffusing through the membrane lipid.

Anion-dependent Mg2+ influx and a role for a vacuolar H+-ATPase in sheep ruminal epithelial cells

2003 ◽  
Vol 285 (1) ◽  
pp. G45-G53 ◽  
Author(s):  
Monika Schweigel ◽  
Holger Martens
Keyword(s):  
Epithelial Cells ◽  
Atpase Activity ◽  
Primary Culture ◽  
Intracellular Ph ◽  
Uptake Rate ◽  
Ruminal Fermentation ◽  
Fermentation Products ◽  
Fluorescence Techniques ◽  
Transport Inhibitors ◽  
Stimulation Of

The K+-insensitive component of Mg2+ influx in primary culture of ruminal epithelial cells (REC) was examined by means of fluorescence techniques. The effects of extracellular anions, ruminal fermentation products, and transport inhibitors on the intracellular free Mg2+ concentration ([Mg2+]i), Mg2+ uptake, and intracellular pH were determined. Under control conditions (HEPES-buffered high-NaCl medium), the [Mg2+]i of REC increased from 0.56 ± 0.14 to 0.76 ± 0.06 mM, corresponding to a Mg2+ uptake rate of 15 μM/min. Exposure to butyrate did not affect Mg2+ uptake, but it was stimulated (by 84 ± 19%) in the presence of [Formula: see text]. In contrast, Mg2+ uptake was strongly diminished if REC were suspended in [Formula: see text]-buffered high-KCl medium (22.3 ± 4 μM/min) rather than in HEPES-buffered KCl medium (37.5 ± 6 μM/min). After switching from high- to low-Cl– solution, [Mg2+]i was reduced from 0.64 ± 0.09 to 0.32 ± 0.16 mM and the [Formula: see text]-stimulated Mg2+ uptake was completely inhibited. Bumetanide and furosemide blocked the rate of Mg2+ uptake by 64 and 40%, respectively. Specific blockers of vacuolar H+-ATPase reduced the [Mg2+]i (36%) and Mg2+ influx (38%) into REC. We interpret this data to mean that the K+-insensitive Mg2+ influx into REC is mediated by a cotransport of Mg2+ and Cl– and is energized by an H+-ATPase. The stimulation of Mg2+ transport by ruminal fermentation products may result from a modulation of the H+-ATPase activity.

Opposite effects of Ni2+ on Xenopus and rat ENaCs expressed in Xenopus oocytes

2005 ◽  
Vol 289 (4) ◽  
pp. C946-C958 ◽  
Author(s):  
Dana Cucu ◽  
Jeannine Simaels ◽  
Jan Eggermont ◽  
Willy Van Driessche ◽  
Wolfgang Zeiske
Keyword(s):  
Voltage Clamp ◽  
Serine Proteases ◽  
Metal Ion ◽  
Xenopus Oocytes ◽  
Noise Analysis ◽  
Inhibitory Effect ◽  
Rat Colon ◽  
Current Response ◽  
Α Subunit ◽  
Kinetics Of

Opposite effects of Ni2+ on Xenopus and rat ENaCs expressed in Xenopus oocytes. Am J Physiol Cell Physiol 289: C946–C958, 2005. First published June 8, 2005; .—The epithelial Na+ channel (ENaC) is modulated by various extracellular factors, including Na+, organic or inorganic cations, and serine proteases. To identify the effect of the divalent Ni2+ cation on ENaCs, we compared the Na+ permeability and amiloride kinetics of Xenopus ENaCs (xENaCs) and rat ENaCs (rENaCs) heterologously expressed in Xenopus oocytes. We found that the channel cloned from the kidney of the clawed toad Xenopus laevis [wild-type (WT) xENaC] was stimulated by external Ni2+, whereas the divalent cation inhibited the channel cloned from the rat colon (WT rENaC). The kinetics of amiloride binding were determined using noise analysis of blocker-induced fluctuation in current adapted for the transoocyte voltage-clamp method, and Na+ conductance was assessed using the dual electrode voltage-clamp (TEVC) technique. The inhibitory effect of Ni2+ on amiloride binding is not species dependent, because Ni2+ decreased the affinity (mainly reducing the association rate constant) of the blocker in both species in competition with Na+. Importantly, using the TEVC method, we found a prominent difference in channel conductance at hyperpolarizing voltage pulses. In WT xENaCs, the initial ohmic current response was stimulated by Ni2+, whereas the secondary voltage-activated current component remained unaffected. In WT rENaCs, only a voltage-dependent block by Ni2+ was obtained. To further study the origin of the xENaC stimulation by Ni2+, and based on the rationale of the well-known high affinity of Ni2+ for histidine residues, we designed α-subunit mutants of xENaCs by substituting histidines that were expressed in oocytes, together with WT β- and γ-subunits. Changing His215 to Asp in one putative amiloride-binding domain (WYRFHY) in the extracellular loop between Na+ channel membrane segments M1 and M2 had no influence on the stimulatory effect of Ni2+, and neither did complete deletion of this segment. Next, we mutated His416 flanked by His411 and Cys417, a unique site for possible heavy metal ion chelation, and, with this quality, most proximal (∼100 amino acids upstream of the second putative amiloride binding site at the pore entrance), was found localized at M2. Replacing His416 with arginine, aspartate, tyrosine, and alanine clearly affected amiloride binding in all cases, as well as Na+ conductance, as expressed in the xENaC current-voltage relationship, especially with regard to aspartate and tyrosine. However, similarly to those obtained with the WYRFHY stretch, none of these mutations could either abolish the stimulating effect of Ni2+ or reverse it to an inhibitory type.

scholarly journals Towards Understanding the Involvement of H+-ATPase in Programmed Cell Death of Psammosilene tunicoides after Oxalic Acid Application

Molecules ◽  
2021 ◽  
Vol 26 (22) ◽  
pp. 6957
Author(s):  
Xinyu Jiang ◽  
Mohammad Aqa Mohammadi ◽  
Yuan Qin ◽  
Zongshen Zhang
Keyword(s):  
Cell Death ◽  
Programmed Cell Death ◽  
Oxalic Acid ◽  
Atpase Activity ◽  
Intracellular Ph ◽  
Death Rate ◽  
Ph Value ◽  
Intracellular Acidification ◽  
Cell Death Rate

Psammosilene tunicoides is a unique perennial medicinal plant species native to the Southwestern regions of China. Its wild population is rare and endangered due to over-excessive collection and extended growth (4–5 years). This research shows that H+-ATPase activity was a key factor for oxalate-inducing programmed cell death (PCD) of P. tunicoides suspension cells. Oxalic acid (OA) is an effective abiotic elicitor that enhances a plant cell’s resistance to environmental stress. However, the role of OA in this process remains to be mechanistically unveiled. The present study evaluated the role of OA-induced cell death using an inverted fluorescence microscope after staining with Evans blue, FDA, PI, and Rd123. OA-stimulated changes in K+ and Ca2+ trans-membrane flows using a patch-clamp method, together with OA modulation of H+-ATPase activity, were further examined. OA treatment increased cell death rate in a dosage-and duration-dependent manner. OA significantly decreased the mitochondria activity and damaged its electron transport chain. The OA treatment also decreased intracellular pH, while the FC increased the pH value. Simultaneously, NH4Cl caused intracellular acidification. The OA treatment independently resulted in 90% and the FC led to 25% cell death rates. Consistently, the combined treatments caused a 31% cell death rate. Furthermore, treatment with EGTA caused a similar change in intracellular pH value to the La3+ and OA application. Combined results suggest that OA-caused cell death could be attributed to intracellular acidification and the involvement of OA in the influx of extracellular Ca2+, thereby leading to membrane depolarization. Here we explore the resistance mechanism of P. tunicoides cells against various stresses endowed by OA treatment.

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