Intracellular Proton Concentration Research Articles

Inhibition of NHE1 transport activity and gene transcription in DRG neurons in oxaliplatin-induced painful peripheral neurotoxicity

Oxaliplatin (OHP)-induced peripheral neurotoxicity (OIPN), one of the major dose-limiting side effects of colorectal cancer treatment, is characterized by both acute and chronic syndromes. Acute exposure to low dose OHP on dorsal root ganglion (DRG) neurons is able to induce an increase in intracellular calcium and proton concentration, thus influencing ion channels activity and neuronal excitability. The Na+/H+ exchanger isoform-1 (NHE1) is a plasma membrane protein that plays a pivotal role in intracellular pH (pHi) homeostasis in many cell types, including nociceptors. Here we show that OHP has early effects on NHE1 activity in cultured mouse DRG neurons: the mean rate of pHi recovery was strongly reduced compared to vehicle-treated controls, reaching levels similar to those obtained in the presence of cariporide (Car), a specific NHE1 antagonist. The effect of OHP on NHE1 activity was sensitive to FK506, a specific calcineurin (CaN) inhibitor. Lastly, molecular analyses revealed transcriptional downregulation of NHE1 both in vitro, in mouse primary DRG neurons, and in vivo, in an OIPN rat model. Altogether, these data suggest that OHP-induced intracellular acidification of DRG neurons largely depends on CaN-mediated NHE1 inhibition, revealing new mechanisms that OHP could exert to alter neuronal excitability, and providing novel druggable targets.

Modeling of pH regulation in tumor cells: Direct interaction between proton-coupled lactate transporters and cancer-associated carbonic anhydrase.

The most aggressive tumor cells, which often reside in a hypoxic environment, can release vast amounts of lactate and protons via monocarboxylate transporters (MCTs). This additional proton efflux exacerbates extracellular acidification and supports the formation of a hostile environment. In the present study we propose a novel, data-based model for this proton-coupled lactate transport in cancer cells. The mathematical settings involve systems coupling nonlinear ordinary and stochastic differential equations describing the dynamics of intra- and extracellular proton and lactate concentrations. The data involve time series of intracellular proton concentrations of normoxic and hypoxic MCF-7 breast cancer cells. The good agreement of our final model with the data suggests the existence of proton pools near the cell membrane, which can be controlled by intracellular and extracellular carbonic anhydrases to drive proton-coupled lactate transport across the plasma membrane of hypoxic cancer cells.

On a coupled SDE-PDE system modeling acid-mediated tumor invasion

We propose and analyze a multiscale model for acid-mediated tumor invasion accounting for stochastic effects on the subcellular level. The setting involves a PDE of reaction-diffusion-taxis type describing the evolution of the tumor cell density, the movement being directed towards pH gradients in the local microenvironment, which is coupled to a PDE-SDE system characterizing the dynamics of extracellular and intracellular proton concentrations, respectively. The global well-posedness of the model is shown and numerical simulations are performed in order to illustrate the solution behavior.

Elucidation of toxicity of organic acids inhibiting growth of Escherichia coli W

The toxic effects of 3-hydroxypropionic acid (3-HP) at high concentrations on cell growth and cellular metabolism are a great challenge to its commercial production. This study has examined and compared the toxic effects of 3-HP on cell growth with other similar weak acids, especially lactic acid, under various concentrations, temperatures and pH using Escherichia coli W as the test strain. 3-HP was approximately 4.4-times more toxic than lactic acid due to the 4.4-fold weaker acidity or 0.64 higher pKa value. The two acids presented no appreciable difference when the growth inhibition was correlated with the undissociated or protonated free acid concentration calculated by the Henderson-Hasselbalch equation. The growth inhibition by other small organic acids, such as acetic acid, pyruvic acid, propionic acid, 2-hydroxybutyric acid (2-HB) and 3-hydroxybutyric acid (3-HB), was also well correlated with their pKa values or protonated free acid concentrations. This study suggests that the growth inhibition by small weak acids is mainly caused by the socalled proton effect (rather than the anion effect), i.e., an increase in the intracellular proton concentration. An appropriate increase in the medium pH was suggested to alleviate the acid toxicity by reducing the free acid concentration in the culture medium.

Keeping It Simple, Transport Mechanism and pH Regulation in Na+/H+ Exchangers

Na(+)/H(+) exchangers are essential for regulation of intracellular proton and sodium concentrations in all living organisms. We examined and experimentally verified a kinetic model for Na(+)/H(+) exchangers, where a single binding site is alternatively occupied by Na(+) or one or two H(+) ions. The proposed transport mechanism inherently down-regulates Na(+)/H(+) exchangers at extreme pH, preventing excessive cytoplasmic acidification or alkalinization. As an experimental test system we present the first electrophysiological investigation of an electroneutral Na(+)/H(+) exchanger, NhaP1 from Methanocaldococcus jannaschii (MjNhaP1), a close homologue of the medically important eukaryotic NHE Na(+)/H(+) exchangers. The kinetic model describes the experimentally observed substrate dependences of MjNhaP1, and the transport mechanism explains alkaline down-regulation of MjNhaP1. Because this model also accounts for acidic down-regulation of the electrogenic NhaA Na(+)/H(+) exchanger from Escherichia coli (EcNhaA, shown in a previous publication) we conclude that it applies generally to all Na(+)/H(+) exchangers, electrogenic as well as electroneutral, and elegantly explains their pH regulation. Furthermore, the electrophysiological analysis allows insight into the electrostatic structure of the translocation complex in electroneutral and electrogenic Na(+)/H(+) exchangers.

Dimer is a Minimal Functional Unit of Influenza a Virus M2 Channel on Living Cells

The integral matrix protein M2 of influenza A virus has been proposed to form an acid-activated, proton-selective channel in the viral lipid envelope upon infecting host cells. Although X-ray crystallography and NMR studies using fragment peptides suggested that M2 stably forms a tetrameric channel irrespective of pH, the oligomeric states of the full-length protein on living cells are not clear yet. In the present study, we investigated the oligomerization of the protein in living CHO cells using the coiled-coil labeling method developed in our laboratory and the spectral imaging technique [1]. Contrary to previous models, the protein existed as a dimer at neutral pH, although it formed a tetramer at pH below 5. The proton channel activity was determined with the pH-sensitive dye SNARF-4F. The intracellular proton concentration was exponentially increased when the extracellular pH was reduced. The rate constant per protein for the dimer was larger than that for the tetramer. Alanine-scanning experiments showed that His37 was crucial to the channel activity. The addition of the antiviral drug amantadine hydrochloride inhibited the tetramerization at acidic pH and the activity of the dimeric channel, suggesting that the drug binds to the dimeric form and blocks both the proton conductance and tetramerization instead of clogging the tetrameric pore. Cholesterol removal resulted in a significant decrease in the activity of the dimer. These results indicate that a minimum functional unit of M2 protein is dimer, which forms a complex with cholesterol for its function.[1] Kawano et al., Anal. Chem. 85, 3454 (2013)

Identification of Organic Acids in Wine That Stimulate Mechanisms of Gastric Acid Secretion

Wine may cause stomach irritation due to its stimulatory effect on gastric acid secretion, although the mechanisms by which wine or components thereof activate pathways of gastric acid secretion are poorly understood. Gastric pH was measured with a noninvasive intragastric probe, demonstrating that administration of 125 mL of white or red wine to healthy volunteers stimulated gastric acid secretion more potently than the administration of equivalent amounts of ethanol. Between both beverages, red wine showed a clear trend for being more active in stimulating gastric acid secretion than white wine (p = 0.054). Quantification of the intracellular proton concentration in human gastric tumor cells (HGT-1), a well-established indicator of proton secretion and, in turn, stomach acid formation in vivo, confirmed the stronger effect of red wine as compared to white wine. RT-qPCR experiments on cells exposed to red wine also revealed a more pronounced effect than white wine on the fold change expression of genes associated with gastric acid secretion. Of the quantitatively abundant organic acids in wine, malic acid and succinic acid most actively stimulated proton secretion in vitro. However, addition of ethanol to individual organic acids attenuated the secretory effect of tartaric acid, but not that of the other organic acids. It was concluded that malic acid for white wine and succinic acid for red wine are key organic acids that contribute to gastric acid stimulation.

Lidocaine depolarizes the mitochondrial membrane potential by intracellular alkalization in rat dorsal root ganglion neurons

The mitochondrial membrane potential (ΔΨm) is an important factor for apoptosis, and it is produced by the proton electrochemical gradient (ΔµH(+)). Therefore, the intracellular proton concentration (pH(in)) is an important factor for modifying the ΔΨm. However, the effects of lidocaine on pH(in) are unclear. To investigate mitochondrial responses to lidocaine, therefore, we simultaneously measured pH(in) with ΔΨm, flavin adenine dinucleotide (FAD), and reduced form of nicotinamide adenine dinucleotide (NADH) fluorescence, and calculated the FAD/NADH ratio (redox ratio), the superoxide production in mitochondria. Morphological change and early apoptosis were observed by annexin-V FITC staining under fluorescent microscope. The ratiometric fluorescent probe JC-1 and HPTS were used for the simultaneous measurements of ΔΨm with pH(in) in rat dorsal root ganglion (DRG) neurons. FAD and NADH autofluorescence were simultaneously measured, and the FAD/NADH fluorescence ratio (redox ratio) was calculated. The superoxide was measured by mitosox-red fluorescent probe for mitochondrial superoxide. Lidocaine was evaluated at 1, 5, and 10mM. Morphological change and early apoptosis were observed after 10mM lidocaine administration. Lidocaine depolarized ΔΨm with increased pH(in) in a dose-dependent manner. In low-pH saline (pH 6), in the presence of both the weak acids (acetate and propionate), lidocaine failed to depolarize ΔΨm and increase pH(in). On the other hand, lidocaine decreased the redox ratio in the cell and increased the levels of superoxide in a dose-dependent manner. These results demonstrated that lidocaine depolarizes ΔΨm by intracellular alkalization. These results may indicate one of the mechanisms responsible for lidocaine-induced neurotoxicity.

A molecular mechanism for proton-dependent gating in KcsA

Activation gating in KcsA is elicited by changes in intracellular proton concentration. Thompson et al. [1] identified a charge cluster around the inner gate that plays a key role in defining proton activation in KcsA. Here, through functional and spectroscopic approaches, we confirmed the role of this charge cluster and now provide a mechanism of pH-dependent gating. Channel opening is driven by a set of electrostatic interactions that include R117, E120 and E118 at the bottom of TM2 and H25 at the end of TM1. We propose that electrostatic compensation in this charge cluster stabilizes the closed conformation at neutral pH and that its disruption at low pH facilitates the transition to the open conformation by means of helix–helix repulsion.

Charge Substitution of a Deep-Pore Residue Suggests Structural Rearrangements During BK Channel Activation

During scanning mutagenesis of the S6 region of BK channels, we found a deep pore residue (M314 in hSlo) with interesting gating phenotypes. At pH7, the histidine substitution at this position (M314H) gave rise to currents with significantly slower deactivation kinetics than the wild-type BK. Deactivation became even slower with intracellular pH6 and pH5. To test whether this observation is an effect due to side-chain charges, we made three other charge-substituting mutants, M314K, M314E and M314D. For M314K, deactivation kinetics of the currents at pH7 was similar to that of M314H at pH5. This is consistent with the fact that the lysine (K) side-chain has a more basic pKa than that of histidine (H). For both M314H and M314K, increasing proton concentration (lowering pH) made the deactivation slower while reducing proton concentration (increasing pH) made the deactivation faster. For M314E, deactivation kinetics of the currents was fast at pH5 and became slower as the intracellular proton concentration was reduced to pH6, 7, 8, 9 and 10. The M314D channels could stay open at negative potentials in 0 Ca2+, at pH7, 8, 9 and 10. The most effective way to close the M314D channels was to increase the intracellular proton concentration to pH5. The results from all the charge-substituted mutants (M314H, M314K, M314E and M314D) support the idea that neutralizing the side-chain at the 314 position helped the channels close while charging the side-chain made it difficult for the channels to close. We propose that the charged form of the substituted residues may prefer the polar environment of the pore and stabilize the open state. The 314 residue may become more exposed to the pore as the channels transition from the closed to the open conformation.

Conversion of the 2 Cl−/1 H+ antiporter ClC-5 in a NO3−/H+ antiporter by a single point mutation

Several members of the CLC family are secondary active anion/proton exchangers, and not passive chloride channels. Among the exchangers, the endosomal ClC-5 protein that is mutated in Dent's disease shows an extreme outward rectification that precludes a precise determination of its transport stoichiometry from measurements of the reversal potential. We developed a novel imaging method to determine the absolute proton flux in Xenopus oocytes from the extracellular proton gradient. We determined a transport stoichiometry of 2 Cl(-)/1 H+. Nitrate uncoupled proton transport but mutating the highly conserved serine 168 to proline, as found in the plant NO3(-)/H+ antiporter atClCa, led to coupled NO3(-)/H+ exchange. Among several amino acids tested at position 168, S168P was unique in mediating highly coupled NO3(-)/H+ exchange. We further found that ClC-5 is strongly stimulated by intracellular protons in an allosteric manner with an apparent pK of approximately 7.2. A 2:1 stoichiometry appears to be a general property of CLC anion/proton exchangers. Serine 168 has an important function in determining anionic specificity of the exchange mechanism.

Effect of Intracellular pH on the Torque–Speed Relationship of Bacterial Proton-Driven Flagellar Motor

Bacterial flagella responsible for motility are driven by rotary motors powered by the electrochemical potential difference of specific ions across the cytoplasmic membrane. The stator of proton-driven flagellar motor converts proton influx into mechanical work. However, the energy conversion mechanism remains unclear. Here, we show that the motor is sensitive to intracellular proton concentration for high-speed rotation at low load, which was considerably impaired by lowering intracellular pH, while zero-speed torque was not affected. The change in extracellular pH did not show any effect. These results suggest that a high intracellular proton concentration decreases the rate of proton translocation and therefore that of the mechanochemical reaction cycle of the motor but not the actual torque generation step within the cycle by the stator–rotor interactions.

In VivoMolecular Imaging of Cervical Neoplasia Using Acetic Acid as Biomarker

In this paper, a molecular imaging method employing acetic acid dilute solution as a biomarker is described. An interpretation of the biophysical processes that are involved in the biomarker-tissue interaction and are determining the in vivo measured dynamic scattering characteristics is presented. On the basis of this interpretation, a compartmental model of the epithelium is developed for predicting the epithelial transport phenomena that are expected to be correlated with the dynamic characteristics of the backscattered light. The model predictions have been compared with the experimental data obtained from patients with cervical neoplasia of different grade, with the aid of a specially developed imaging system. Comparisons confirmed the validity of the interpretation of the phenomenon, and particularly, the fact that dynamic scattering characteristics are largely determined by the intracellular proton concentration kinetics. In addition, the correlation of the latter with both structural and functional alterations, associated with cervical neoplasia development, has been predicted theoretically and confirmed experimentally. The established correlation enables the derivation of quantitative indices expressing disease-specific microstructural and functional alterations, from the in vivo measured dynamic optical characteristics. This highlights the potential of the developed imaging method and technology for the noninvasive diagnosis, guided therapeutics, and screening of cervical neoplasia.

Escherichia coli acid resistance: pH-sensing, activation by chloride and autoinhibition in GadB.

Escherichia coli and other enterobacteria exploit the H+ -consuming reaction catalysed by glutamate decarboxylase to survive the stomach acidity before reaching the intestine. Here we show that chloride, extremely abundant in gastric secretions, is an allosteric activator producing a 10-fold increase in the decarboxylase activity at pH 5.6. Cooperativity and sensitivity to chloride were lost when the N-terminal 14 residues, involved in the formation of two triple-helix bundles, were deleted by mutagenesis. X-ray structures, obtained in the presence of the substrate analogue acetate, identified halide-binding sites at the base of each N-terminal helix, showed how halide binding is responsible for bundle stability and demonstrated that the interconversion between active and inactive forms of the enzyme is a stepwise process. We also discovered an entirely novel structure of the cofactor pyridoxal 5'-phosphate (aldamine) to be responsible for the reversibly inactivated enzyme. Our results link the entry of chloride ions, via the H+/Cl- exchange activities of ClC-ec1, to the trigger of the acid stress response in the cell when the intracellular proton concentration has not yet reached fatal values.

Effect of intracellular pH on rotational speed of bacterial flagellar motors.

Weak acids such as acetate and benzoate, which partially collapse the transmembrane proton gradient, not only mediate pH taxis but also impair the motility of Escherichia coli and Salmonella at an external pH of 5.5. In this study, we examined in more detail the effect of weak acids on motility at various external pH values. A change of external pH over the range 5.0 to 7.8 hardly affected the swimming speed of E. coli cells in the absence of 34 mM potassium acetate. In contrast, the cells decreased their swimming speed significantly as external pH was shifted from pH 7.0 to 5.0 in the presence of 34 mM acetate. The total proton motive force of E. coli cells was not changed greatly by the presence of acetate. We measured the rotational rate of tethered E. coli cells as a function of external pH. Rotational speed decreased rapidly as the external pH was decreased, and at pH 5.0, the motor stopped completely. When the external pH was returned to 7.0, the motor restarted rotating at almost its original level, indicating that high intracellular proton (H+) concentration does not irreversibly abolish flagellar motor function. Both the swimming speeds and rotation rates of tethered cells of Salmonella also decreased considerably when the external pH was shifted from pH 7.0 to 5.5 in the presence of 20 mM benzoate. We propose that the increase in the intracellular proton concentration interferes with the release of protons from the torque-generating units, resulting in slowing or stopping of the motors.

Elevated intracellular zinc and altered proton homeostasis in forebrain neurons

Using the H +-sensitive fluorophore 2′,7′-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein (BCECF) and microfluorimetry, we investigated how elevated intracellular free zinc ([Zn 2+] i) altered intracellular proton concentration (pH i) in dissociated cultures of rat forebrain neurons. Neurons exposed to extracellular zinc (3 μM) in the presence of the Zn 2+-selective ionophore pyrithione (20 μM) underwent intracellular acidification that was not reversed upon washout of the stimulus. Application of a membrane-permeant Zn 2+ chelator, but not an impermeant chelator, partially restored pH i. Removal of extracellular Ca 2+ greatly inhibited [Zn 2+] i-induced acidification, suggesting that acidification was a secondary consequence of Ca 2+ entry. Additional experiments suggested that Ca 2+ entered through the plasma membrane sodium/calcium exchanger (NCE), because a specific inhibitor of reverse mode NCE operation, KB-R7943 (1 μM), significantly inhibited Zn 2+-induced acidification. In addition to the phenomenon of [Zn 2+] i-induced acidification, we found that elevated [Zn 2+] i inhibited neuronal recovery from low pH i. Neurons exposed to a protonophore underwent robust acidification, and pH i recovery ensued upon protonophore washout. In contrast, neurons acidified by the protonophore in the presence of Zn 2+ (3 μM) and pyrithione (20 μM) showed no ability to recover from low pH i. Application of a membrane-permeant Zn 2+ chelator partially restored pH i to pre-stimulus values. Experiments designed to elucidate mechanisms responsible for pH i regulation revealed that neurons relied primarily on bicarbonate exchange for proton export, suggesting that elevated [Zn 2+] i might impede pH i by inhibiting proton efflux via bicarbonate exchange. These results provide novel insights into the physiological effects of raising [Zn 2+] i, and may help illuminate the mechanisms by which Zn 2+ injures neurons.

Acute inhibition of brain-specific Na(+)/H(+) exchanger isoform 5 by protein kinases A and C and cell shrinkage.

Little is known of the functional properties of the mammalian, brain-specific Na(+)/H(+) exchanger isoform 5 (NHE5). Rat NHE5 was stably expressed in NHE-deficient PS120 cells, and its activity was characterized using the fluorescent pH-sensitive dye 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein. NHE5 was insensitive to ethylisopropyl amiloride. The transport kinetics displayed a simple Michaelis-Menten relationship for extracellular Na(+) (apparent K(Na) = 27 +/- 5 mM) and a Hill coefficient near 3 for the intracellular proton concentration with a half-maximal activity at an intracellular pH of 6.93 +/- 0.03. NHE5 activity was inhibited by acute exposure to 8-bromo-cAMP or forskolin (which increases intracellular cAMP by activating adenylate cyclase). The kinase inhibitor H-89 reversed this inhibition, suggesting that regulation by cAMP involves a protein kinase A (PKA)-dependent process. In contrast, 8-bromo-cGMP did not have a significant effect on activity. The protein kinase C (PKC) activator phorbol 12-myristrate 13-acetate inhibited NHE5, and the PKC antagonist chelerythrine chloride blunted this effect. Activity was also inhibited by hyperosmotic-induced cell shrinkage but was unaffected by a hyposmotic challenge. These results demonstrate that rat brain NHE5 is downregulated by activation of PKA and PKC and by cell shrinkage, important regulators of neuronal cell function.

Protons Inhibit the BKCa Channel of Rat Small Artery Smooth Muscle Cells

The regulation of the activity of calcium-activated potassium (BK_Ca) channels by intracellular proton ions (pH_i) was investigated using the patch-clamp technique in smooth muscle cells freshly isolated from rat tail small arteries. Single-channel conductance and voltage dependence of activation were not different at pH_i 7.0, 7.4 and 7.8. However, the membrane potential at which channel open probability reached 0.5 was 74 ± 5 mV (n = 6) (mean ± SE) at pH_i 7.4 and 54 ± 2 mV (n = 4) at pH_i 7.8 under conditions of pCa 5.9, and 30 ± 5 mV (n = 5) at pH_i 7.4 and 62 ± 4 mV (n = 5) at pH_i 7.0 under conditions of pCa 5.4. Furthermore, at a membrane potential of 0 mV, the pD₂ for intracellular calcium ions was 5.19 ± 0.04 (n = 26) (mean ± SD) at pH_i 7.8, 5.02 ± 0.05 (n = 28) at pH_i 7.4, and 4.82 ± 0.05 (n = 30) at pH_i 7.0. In addition, an alteration of pH_i resulted in a profound change in the amplitude of BK_Ca currents in intact cells; it reversibly attenuated the current-voltage relationship decreasing the current by 55 ± 3% (n = 7) (p < 0.001) at 70 mV after lowering the extracellular NH₄Cl concentration to decrease the calculated pH_i from 7.2 to 6.8. Thus, alterations of pH_i in the range from 7.0 to 7.8 did not affect single-channel conductance and voltage dependence of activation but markedly altered single BK_Ca channel activity as well as intact cell BK_Ca current amplitude, where an increase of the intracellular proton concentration inhibited this channel.

Fluorimetric multiparameter cell assay at the single cell level fabricated by optical tweezers

A fluorimetric multi-parameter cell sensor at the single cell level is presented which makes it possible to observe the physiological behavior of different cell lines, different physiological parameters, and statistical data at the same time. Different cell types were immobilized at predefined positions with high accuracy using optical tweezers and adhesion promoting surface layers. The process is applicable to both adherent and non-adherent cells. Coating of the immobilization area with mussel adhesive protein was shown to be essential for the process. Intracellular proton and calcium concentrations in different cell classes were simultaneously imaged and the specific activation of T lymphocytes was demonstrated. This method should be especially useful for drug screening due to the small sample volume and high information density.

Chloride-dependent transport of NH4+ into bee retinal glial cells.

Mammalian astrocytes convert glutamate to glutamine and bee retinal glial cells convert pyruvate to alanine. To maintain such amination reactions these glial cells may take up NH4+/NH3. We have studied the entry of NH4+/NH3 into bundles of glial cells isolated from bee retina by using the fluorescent dye BCECF to measure pH. Ammonium caused intracellular pH to decrease by a saturable process: the rate of change of pH was maximal for an ammonium concentration of about 5 mM. This acidifying response to ammonium was abolished by the loop diuretic bumetanide (100 microM) and by removal of extracellular Cl-. These results strongly suggest that ammonium enters the cell by contransport of NH4+ with Cl-. Removal of extracellular Na+ did not abolish the NH(4+)-induced acidification. The NH(4+)-induced pH change was unaffected when nearly all K+ conductance was blocked with 5 mM Ba2+ showing that NH4+ did not enter through Ba(2+)-sensitive ion channels. Application of 2 mM NH4+ led to a large increase in total intracellular proton concentration estimated to exceed 13.5 mEq/L. As the cell membrane appeared to be permeable to NH3, we suggest that when NH4+ entered the cells, NH3 left, so that protons were shuttled into the cell. This shuttle, which was strongly dependent on internal and external pH, was quantitatively modelled. In retinal slices, 2 mM NH4+ alkalinized the extracellular space: this alkalinization was reduced in the absence of bath Cl-. We conclude that NH4+ enters the glial cells in bee retina on a cotransporter with functional similarities to the NH4+(K+)-Cl- cotransporter described in kidney cells.