Kinetic Dissection of Two Distinct Proton Binding Sites in Na+/H+ Exchangers by Measurement of Reverse Mode Reaction

Wakabayashi, Shigeo; Hisamitsu, Takashi; Pang, Tianxiang; Shigekawa, Munekazu

doi:10.1074/jbc.m306690200

Cited by 79 publications

(82 citation statements)

References 37 publications

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“…Based on its expression in Malpighian tubules, AeNHE8 would be expected to function in the forward mode extruding Na ϩ from principal cells into the lumen. But NHEs have been known to function in reverse mode under sufficiently strong electrochemical gradient (54). We developed stable PS120 cell lines expressing tagged and untagged AeNHE8 and confirmed the protein's expression in the plasma membrane using both anti-NHE8 and anti-c-Myc epitope antibodies (Figs.…”

Section: Discussionmentioning

confidence: 99%

NHE8 mediates amiloride-sensitive Na⁺/H⁺exchange across mosquito Malpighian tubules and catalyzes Na⁺and K⁺transport in reconstituted proteoliposomes

Kang'ethe

Aimanova²,

Pullikuth

et al. 2007

American Journal of Physiology-Renal Physiology

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Following a blood meal, the mosquito Aedes aegypti will have acquired an enormous sodium load that must be rapidly excreted to restore ion homeostasis. It is a process that demands robust sodium and fluid transport capabilities. Even though the identities of the components involved in this ion transport across the mosquito Malpighian tubule epithelia have not been completely determined, electrophysiological studies suggest the contribution of a Na+/H+exchanger extruding cations into the lumen driven secondarily by the proton gradient created by the V-type H+-ATPase in the tubules' apical membrane. We have identified the putative exchanger and designated it AeNHE8. Immunolocalization studies demonstrated that AeNHE8 is expressed in the apical membranes of Malpighian tubules, gastric caecae, and rectum. When heterologously expressed in salt-sensitive yeast cells lacking Na+extrusion and Na+/H+exchange proteins, AeNHE8 rescues the salt-sensitive phenotype and restores the cells' ability to grow in high NaCl media. Furthermore, heterologous expression of AeNHE8 in NHE-deficient fibroblast cells results in an amiloride-sensitive22Na+uptake. To determine the exchanger's kinetic properties, we reconstituted membranes from yeast cells expressing the protein into lipid proteoliposomes and assayed for cation-dependent H+exchange by fluorimetric methods. Our results indicate that AeNHE8 mediates saturable exchange of Na+and K+for H+. We propose that AeNHE8 may be coupled to the inward H+gradient across the Malpighian tubules and plays a role in the extrusion of excess sodium and potassium while maintaining steady intracellular pH in the principal cells.

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Section: Discussionmentioning

confidence: 99%

NHE8 mediates amiloride-sensitive Na⁺/H⁺exchange across mosquito Malpighian tubules and catalyzes Na⁺and K⁺transport in reconstituted proteoliposomes

Kang'ethe

Aimanova²,

Pullikuth

et al. 2007

American Journal of Physiology-Renal Physiology

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“…7, obtained in ATP-depleted parasites. The postulated shift of the pH i dependence curve under ATP-depleted conditions, away from the normal resting pH i , mirrors the behavior of the mammalian Na ϩ /H ϩ exchanger (26). The black circle indicates the activity of the system in ATP-replete parasites at the normal resting pH i (ϳ7.3).…”

Section: The Flux Of H ϩ -Equivalents Following the Removal And Restomentioning

confidence: 99%

“…14C) that the pH i dependence of the system, like that of the mammalian Na ϩ /H ϩ exchanger (26), shifts under conditions of ATP depletion. For the mammalian Na ϩ /H ϩ exchanger, ATP depletion causes the curve describing the pH i dependence of the exchanger to shift such that a larger perturbation of pH i is required to activate the system than is the case under ATP-replete conditions (26). If the same were true for the system described here then, as illustrated schematically in Fig.…”

mentioning

confidence: 99%

An Acid-loading Chloride Transport Pathway in the Intraerythrocytic Malaria Parasite, Plasmodium falciparum

Henry

Cobbold

Khan

et al. 2010

Journal of Biological Chemistry

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“…4) are similar to those in corresponding studies in which the stimulatory effect of these fermentation products on amiloride-sensitive Na ϩ absorption via NHE has been characterized (24,26,53,54,77). As in monogastric animals in which Na ϩ uptake also increases in response to a change in the luminal end products of digestion (20), the underlying transporter is NHE3, an extremely well-characterized transport protein that utilizes the energy from the influx of Na ϩ to drive the efflux of H ϩ on a 1:1 basis, in allosterically regulated manner so that the extrusion of protons stops when the cytosolic pH reaches the set point optimal for cellular function (3,20,91).…”

Section: Na and Urea Transportmentioning

confidence: 99%

Modulation of urea transport across sheep rumen epithelium in vitro by SCFA and CO₂

Abdoun

Stumpff

Rabbani

et al. 2010

American Journal of Physiology-Gastrointestinal and Liver Physi

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Urea transport across the gastrointestinal tract involves transporters of the urea transporter-B group, the regulation of which is poorly understood. The classical stimulatory effect of CO2 and the effect of short-chain fatty acids (SCFA) on the ruminal recycling of urea were investigated by using Ussing chamber and microelectrode techniques with isolated ruminal epithelium of sheep. The flux of urea was found to be phloretin sensitive and passive. At a luminal pH of 6.4, but not at 7.4, the addition of SCFA (40 mmol/l) or CO2/HCO 3 Ϫ (10% and 25 mmol/l) led to a fourfold increase in urea flux. The stepwise reduction of luminal pH in the presence of SCFA from 7.4 to 5.4 led to a bell-shaped modification of urea transport, with a maximum at pH 6.2. Lowering the pH in the absence of SCFA or CO2 had no effect. Inhibition of Na ϩ /H ϩ exchange increased urea flux at pH 7.4, with a decrease being seen at pH 6.4. In experiments with double-barreled, pH-sensitive microelectrodes, we confirmed the presence of an apical pH microclimate and demonstrated the acidifying effects of SCFA on the underlying epithelium. We confirm that the permeability of the ruminal epithelium to urea involves a phloretin-sensitive pathway. We present clear evidence for the regulation of urea transport by strategies that alter intracellular pH, with permeability being highest after a moderate decrease. The well-known postprandial stimulation of urea transport to the rumen in vivo may involve acute pH-dependent effects of intraruminal SCFA and CO2 on the function of existing urea transporters. pH i; urea transporter-B; short-chain fatty acids; microclimate; volatile fatty acid UREA, POSSIBLY BECAUSE OF its small size, was long thought to move passively across epithelia, depending only on the rate of delivery via blood. The urea permeability of cellular membranes has now been established to be several orders of magnitude above that of lipid membranes (11,92) and is coupled to the expression of specific urea-transporting proteins with channel-like kinetics (7,46,71,81,82,95). Whereas the role that these proteins play in the elegant renal concentrating mechanism has received much attention, their function and regulation in other parts of the body, such as the gut (39), continues to be poorly understood.In contrast to the paucity of our knowledge concerning extrarenal urea transport in humans, we have long known of the ability of camels, cows, or sheep to shift the excretion of urea from the kidney (62, 72) to the gastrointestinal tract (79). The transport of urea through the rumen epithelium was first demonstrated many years ago in vivo and in vitro (16,29,79,89) and the physiological significance is clear: in the rumen, dietary cellulose is broken down by bacteria that utilize ureanitrogen for the synthesis of microbial proteins. After passage into the duodenum, the amino acids of these proteins are absorbed and reach the liver, where new urea for secretion into the rumen can be formed. Recycling of nitrogen via urea secretion into the rumen thus ...

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Kinetic Dissection of Two Distinct Proton Binding Sites in Na+/H+ Exchangers by Measurement of Reverse Mode Reaction

Cited by 79 publications

References 37 publications

NHE8 mediates amiloride-sensitive Na⁺/H⁺exchange across mosquito Malpighian tubules and catalyzes Na⁺and K⁺transport in reconstituted proteoliposomes

NHE8 mediates amiloride-sensitive Na⁺/H⁺exchange across mosquito Malpighian tubules and catalyzes Na⁺and K⁺transport in reconstituted proteoliposomes

An Acid-loading Chloride Transport Pathway in the Intraerythrocytic Malaria Parasite, Plasmodium falciparum

Modulation of urea transport across sheep rumen epithelium in vitro by SCFA and CO₂

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Kinetic Dissection of Two Distinct Proton Binding Sites in Na+/H+ Exchangers by Measurement of Reverse Mode Reaction

Cited by 79 publications

References 37 publications

NHE8 mediates amiloride-sensitive Na+/H+exchange across mosquito Malpighian tubules and catalyzes Na+and K+transport in reconstituted proteoliposomes

NHE8 mediates amiloride-sensitive Na+/H+exchange across mosquito Malpighian tubules and catalyzes Na+and K+transport in reconstituted proteoliposomes

An Acid-loading Chloride Transport Pathway in the Intraerythrocytic Malaria Parasite, Plasmodium falciparum

Modulation of urea transport across sheep rumen epithelium in vitro by SCFA and CO2

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NHE8 mediates amiloride-sensitive Na⁺/H⁺exchange across mosquito Malpighian tubules and catalyzes Na⁺and K⁺transport in reconstituted proteoliposomes

NHE8 mediates amiloride-sensitive Na⁺/H⁺exchange across mosquito Malpighian tubules and catalyzes Na⁺and K⁺transport in reconstituted proteoliposomes

Modulation of urea transport across sheep rumen epithelium in vitro by SCFA and CO₂