Acidic pHi Research Articles

Glucose Uptake and Intracellular pH in a Mouse Model of Ductal Carcinoma In situ (DCIS) Suggests Metabolic Heterogeneity.

Mechanisms for the progression of ductal carcinoma in situ (DCIS) to invasive breast carcinoma remain unclear. Previously we showed that the transition to invasiveness in the mammary intraepithelial neoplastic outgrowth (MINO) model of DCIS does not correlate with its serial acquisition of genetic mutations. We hypothesized instead that progression to invasiveness depends on a change in the microenvironment and that precancer cells might create a more tumor-permissive microenvironment secondary to changes in glucose uptake and metabolism. Immunostaining for glucose transporter 1 (GLUT1) and the hypoxia marker carbonic anhydrase 9 (CAIX) in tumor, normal mammary gland and MINO (precancer) tissue showed differences in expression. The uptake of the fluorescent glucose analog dye, 2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl) amino]-2-deoxy-D-glucose (2-NBDG), reflected differences in the cellular distributions of glucose uptake in normal mammary epithelial cells (nMEC), MINO, and Met1 cancer cells, with a broad distribution in the MINO population. The intracellular pH (pHi) measured using the fluorescent ratio dye 2′,7′-bis(2-carboxyethyl)-5(6)-155 carboxyfluorescein (BCECF) revealed expected differences between normal and cancer cells (low and high, respectively), and a mixed distribution in the MINO cells, with a subset of cells in the MINO having an increased rate of acidification when proton efflux was inhibited. Invasive tumor cells had a more alkaline baseline pHi with high rates of proton production coupled with higher rates of proton export, compared with nMEC. MINO cells displayed considerable variation in baseline pHi that separated into two distinct populations: MINO high and MINO low. MINO high had a noticeably higher mean acidification rate compared with nMEC, but relatively high baseline pHi similar to tumor cells. MINO low cells also had an increased acidification rate compared with nMEC, but with a more acidic pHi similar to nMEC. These findings demonstrate that MINO is heterogeneous with respect to intracellular pH regulation which may be associated with an acidified regional microenvironment. A change in the pH of the microenvironment might contribute to a tumor-permissive or tumor-promoting progression. We are not aware of any previous work showing that a sub-population of cells in in situ precancer exhibits a higher than normal proton production and export rate.

The cardiac electrogenic sodium/bicarbonate cotransporter (NBCe1) is activated by aldosterone through the G protein-coupled receptor 30 (GPR 30)

ABSTRACTThe sodium/bicarbonate cotransporter (NBC) transports extracellular Na+ and HCO3− into the cytoplasm upon intracellular acidosis, restoring the acidic pHi to near neutral values. Two different NBC isoforms have been described in the heart, the electroneutral NBCn1 (1Na+:1HCO3−) and the electrogenic NBCe1 (1Na+:2HCO3−). Certain non-genomic effects of aldosterone (Ald) were due to an orphan G protein-couple receptor 30 (GPR30). We have recently demonstrated that Ald activates GPR30 in adult rat ventricular myocytes, which transactivates the epidermal growth factor receptor (EGFR) and in turn triggers a reactive oxygen species (ROS)- and PI3K/AKT-dependent pathway, leading to the stimulation of NBC. The aim of this study was to investigate the NBC isoform involved in the Ald/GPR30-induced NBC activation. Using specific NBCe1 inhibitory antibodies (a-L3) we demonstrated that Ald does not affect NBCn1 activity. Ald was able to increase NBCe1 activity recorded in isolation. Using immunofluorescence and confocal microscopy analysis we showed in this work that both NBCe1 and GPR30 are localized in t-tubules. In conclusion, we have demonstrated that NBCe1 is the NBC isoform activated by Ald in the heart.

Combination of lactate calcium salt with 5-indanesulfonamide and α-cyano-4-hydroxycinnamic acid to enhance the antitumor effect on HCT116 cells via intracellular acidification.

Maintenance of a neutral intracellular pH (pHi) is favorable for the survival of tumors, and maintenance of highly acidic extracellular pH (pHe) facilitates tumor invasiveness. The aim of the present study was to investigate the antitumor effects of lactate calcium salt (CaLa), 5-indanesulfonamide (IS) and α-cyano-4-hydroxycinnamic acid (CA) via pH regulation in colon cancer cells. HCT116 cells were treated with CaLa, IS, CA and combinations of the three. Subsequently, the concentration of intracellular lactate was determined. pHi and pHe were measured using cell lysates and culture media. Colony formation assay, cell viability assay and western blot analysis were additionally performed to analyze the consequences of the pH changes. CaLa, IS, CA and combination treatments induced an increase in the concentration of intracellular lactate. Lactate influx into the tumor microenvironment produced an acidic pHi in colon cancer cells. Consequently, colony formation and cell viability were significantly decreased, as well as poly(adenosine diphosphate-ribose) polymerase degradation. The tumor microenvironment may be exploited therapeutically by disrupting the mechanism that regulates pHi, leading to cell apoptosis. The present study indicated that treatment with CaLa, IS and CA induced intracellular acidification via lactate influx, causing apoptosis of colon cancer cells. Additionally, the findings suggested that the combination of CaLa with IS and CA may enhance antitumor activity, and may provide a potential therapeutic approach for the treatment of colon cancer.

Identification of Functionally Distinct Na-HCO3 Co-Transporters in Colon

Na-HCO3 cotransport (NBC) regulates intracellular pH (pHi) and HCO3 secretion in rat colon. NBC has been characterized as a 5,5′-diisothiocyanato-2-2′-stilbene (DIDS)-sensitive transporter in several tissues, while the colonic NBC is sensitive to both amiloride and DIDS. In addition, the colonic NBC has been identified as critical for pHi regulation as it is activated by intravesicular acid pH. Molecular studies have identified several characteristically distinct NBC isoforms [i.e. electrogenic (NBCe) and electroneutral (NBCn)] that exhibit tissue specific expression. This study was initiated to establish the molecular identity and specific function of NBC isoforms in rat colon. Northern blot and reverse transcriptase PCR (RT-PCR) analyses revealed that electrogenic NBCe1B or NBCe1C (NBCe1B/C) isoform is predominantly expressed in proximal colon, while electroneutral NBCn1C or NBCn1D (NBCn1C/D) is expressed in both proximal and distal colon. Functional analyses revealed that amiloride-insensitive, electrogenic, pH gradient-dependent NBC activity is present only in basolateral membranes of proximal colon. In contrast, amiloride-sensitive, electroneutral, [H+]-dependent NBC activity is present in both proximal and distal colon. Both electrogenic and electroneutral NBC activities are saturable processes with an apparent Km for Na of 7.3 and 4.3 mM, respectively; and are DIDS-sensitive with apparent Ki of 8.9 and 263.8 µM, respectively. In addition to Na-H exchanger isoform-1 (NHE1), pHi acidification is regulated by a HCO3-dependent mechanism that is HOE694-insensitive in colonic crypt glands. We conclude from these data that electroneutral, amiloride-sensitive NBC is encoded by NBCn1C/D and is present in both proximal and distal colon, while NBCe1B/C encodes electrogenic, amiloride-insensitive Na-HCO3 cotransport in proximal colon. We also conclude that NBCn1C/D regulates HCO3-dependent HOE694-insensitive Na-HCO3 cotransport and plays a critical role in pHi regulation in colonic epithelial cells.

Removal of C.I.Basic yellow 2 from aqueous solution by adsorption onto granular activated carbon using an on-line spectrophotometric analysis system: Kinetic and equilibrium study

The adsorption of Basic yellow 2 (BY2), from aqueous solutions onto Granular activated carbon (GAC) and its adsorption kinetics at different concentrations, GAC masses, initial pH values and temperatures were studied. The adsorption process was followed by an on-line spectrophotometric analysis system, which consisted of UV-spectrophotometer, a designed spectrophotometric cell, peristaltic pump and a glassy reactor. The effect of initial dye concentration, mass of GAC, initial pH and temperature on adsorption was studied over 60 min adsorption period. The results showed that at first the removal percent of BY2 increases with the increase in initial concentration, then the trend is reversed. The results also showed that removal percent of BY2 at pHi around 7 is more than acidic pHi. Also the removal percent of BY2 increases with the increase in mass of GAC and temperature. For kinetic study, the obtained data were treated according to various kinetic models. The results showed that first order kinetic model is fitted to experimental data better than second order and intraparticle diffusion models. The Freundlich, Langmuir, Redlich-Peterson and BET adsorption models were applied to describe the equilibrium isotherms. Type 1 Langmuir isotherm and non-linear Redlich-Peterson isotherm seemed to be the best-fitting models for the experimental results with similar values of coefficient of determination.

Contribution of Intracellular Calcium and pH in Ischemic Uncoupling of Cardiac Gap Junction Channels Formed of Connexins 43, 40, and 45: A Critical Function of C-Terminal Domain

Ischemia is known to inhibit gap junction (GJ) mediated intercellular communication. However the detail mechanisms of this inhibition are largely unknown. In the present study, we determined the vulnerability of different cardiac GJ channels formed of connexins (Cxs) 43, 40, and 45 to simulated ischemia, by creating oxygen glucose deprived (OGD) condition. 5 minutes of OGD decreased the junctional conductance (Gj) of Cx43, Cx40 and Cx45 by 53±3%, 64±1% and 85±2% respectively. Reduction of Gj was prevented completely by restricting the change of both intracellular calcium ([Ca2+]i) and pH (pHi) with potassium phosphate buffer. Clamping of either [Ca2+]i or pHi, through BAPTA (2 mM) or HEPES (80 mM) respectively, offered partial resistance to ischemic uncoupling. Anti-calmodulin antibody attenuated the uncoupling of Cx43 and Cx45 significantly but not of Cx40. Furthermore, OGD could reduce only 26±2% of Gj in C-terminus (CT) truncated Cx43 (Cx43-Δ257). Tethering CT of Cx43 to the CT-truncated Cx40 (Cx40-Δ249), and Cx45 (Cx45-Δ272) helped to resist OGD mediated uncoupling. Moreover, CT domain played a significant role in determining the junction current density and plaque diameter. Our results suggest; OGD mediated uncoupling of GJ channels is primarily due to elevated [Ca2+]i and acidic pHi, though the latter contributes more. Among Cx43, Cx40 and Cx45, Cx43 is the most resistant to OGD while Cx45 is the most sensitive one. CT of Cx43 has major necessary elements for OGD induced uncoupling and it can complement CT of Cx40 and Cx45.

Contribution of calpains to myocardial ischaemia/reperfusion injury

Loss of calcium (Ca(2+)) homeostasis contributes through different mechanisms to cell death occurring during the first minutes of reperfusion. One of them is an unregulated activation of a variety of Ca(2+)-dependent enzymes, including the non-lysosomal cysteine proteases known as calpains. This review analyses the involvement of the calpain family in reperfusion-induced cardiomyocyte death. Calpains remain inactive before reperfusion due to the acidic pHi and increased ionic strength in the ischaemic myocardium. However, inappropriate calpain activation occurs during myocardial reperfusion, and subsequent proteolysis of a wide variety of proteins contributes to the development of contractile dysfunction and necrotic cell death by different mechanisms, including increased membrane fragility, further impairment of Na(+) and Ca(2+) handling, and mitochondrial dysfunction. Recent studies demonstrating that calpain inhibition contributes to the cardioprotective effects of preconditioning and postconditioning, and the beneficial effects obtained with new and more selective calpain inhibitors added at the onset of reperfusion, point to the potential cardioprotective value of therapeutic strategies designed to prevent calpain activation.

Mathematical modeling of the role of carbonic anhydrase II and IV on the influx of CO2 in a Xenopus oocyte

Exposing oocytes to a solution containing CO2/HCO3 causes the familiar fall in intracellular pH (pHi) and a rise in surface pH (pHS) followed by a decay. Musa‐Aziz et al (ASN, 2005) examined the effects of carbonic anhydrases (CAs) on pH transients caused by CO2 influx. They found that injecting CAII into oocytes or expressing CAIV on the oocyte surface accelerates the initial rate of pHi acidification (dpHi/dt) and amplifies the height of the pHS spike (ΔpHS). They proposed that both enzymes enhance CO2 influx by maximizing CO2 gradients across the plasma membrane. We use a mathematical model of a Xenopus oocyte—which accounts for CO2, carbonic acid (H2CO3), HCO3, protons, and a multitude of non‐HCO3 buffer pairs—to investigate the above findings. We simulate the experimental protocol in which the CO2/HCO3 solution is delivered from the bulk to the oocyte surface by raising exponentially the concentrations of CO2, H2CO3 and HCO3 in the bulk and assuming that initially no CO2, H2CO3 and HCO3 are present in the extracellular unconvected fluid. We use the experimental data for 1.5% CO2/10mM HCO3 (pHo=7.5) to find parameter values that simultaneously match ΔpHS, the time that it takes for pHS to reach its peak (tP), dpHi/dt, and the time delay in the pHi decay. We validate the model against data collected when exposing oocytes to 5% CO2/33mM HCO3 and to 10% CO2/66mM HCO3. The model confirms that CAII and CAIV enhance CO2 influx.

Control of Hv1 Voltage-Gated Proton Channel Opening by Changes in the Transmembrane Voltage and pH Gradients

Hv1 proton channels contain a voltage sensor (VS) domain that is homologous to that of tetrameric voltage-gated cation channels (VGCs) and voltage-sensitive phosphatases (VSPs), but cooperative channel gating by changes in both membrane potential (Vm) and the transmembrane pH gradient (ΔpH = pHO - pHI) is apparently unique to Hv1. The mechanism underlying Vm-ΔpH coupling in Hv1, which results in a linear −40 mV/ΔpH unit shift in the GH+-V relation, is not well understood. In order to isolate ΔpH-dependent steps in the Hv1 activation pathway, we measured the effect of changing ΔpH on expressed H+ currents using patch clamp electrophysiology. We show here that Hv1 opening exhibits both voltage-dependent and voltage-independent kinetic components, but closing is purely voltage-dependent. The forward time constant for the final charge-translocating step in Hv1 activation gating is steeply ΔpH-dependent at acidic pHI but only weakly sensitive to ΔpH at alkaline pHI. In addition, we find that at high pHI, a voltage-independent transition becomes rate-limiting for channel opening. Analysis of the relative contributions of voltage-dependent and -independent Hv1 gating components demonstrates that both are necessary to explain the linear Vm-ΔpH relation between pHI 4.5 and 7.5. The effect of changes in ΔpH on Hv1 gating contrasts with Ci-VSP, indicating that Hv1 is particularly well tuned to respond to small perturbations in ΔpH over the physiological range (pHI ≈ pHO). We propose a new multi-state model to account for complex gating in Hv1 in which VS activation and channel opening occur in distinct transitions and display different sensitivities to changes in ΔpH. Vm-ΔpH coupling in Hv1 thus represents an integrated response to changes in both voltage-dependent and -independent gating steps.

Voltage-Dependent Gating of ClC-2 Chloride Channel

The gating of ClC-2 Cl- channel is facilitated by elevated [H+]e and [Cl-]i by interacting with the protopore gate whilst [Cl-]e had not effect. In contrast, the gating of ClC-0 Cl- channel is facilitated by protonation of the protopore gate by intracellular [H+]i in a manner that is dependent on the extracellular [Cl-]e. To gain insights into the Vm dependent mechanism of ClC-2 expressed in HEK cells, we determine the Vm-dependence of open probability (PA(Vm)) at different [H+]i, [H+]e and [Cl-]i using the patch clamp technique. Changing [H+]i by 5 orders of magnitude whilst [Cl-]i/[Cl-]e=140/140 or 10/140 mM did not altered the onset kinetics but channel closing became faster at acidic pHi and PA(Vm) curves were shifted towards more negative Vm. These results suggest that [H+]i did not facilitated gating. In contrast, a same change in [H+]o with [Cl-]i/[Cl-]e=140/140 mM enhanced PA in a bi-phasic manner and shifted PA(Vm) curves to positive Vm. Importantly, PA was >0 with [H+]o=10−10 M and channel closed more slowly when [H+]o or [Cl-]i increased. This implied that ClC-2 can be gated without protonation and that external H+ and/or internal Cl- stabilized the open state. A kinetic analysis of Cl- currents and PA(Vm) curves at different [H+]o and [Cl-]i using a gating scheme coupled to Cl- permeation indicated that protonation of the protopore gate has negligible Vm- and Cl--dependence and that the rate constant for closed-open transition of un-protonated channels were facilitated by elevated [Cl-]i in a Vm-dependent manner. We propose that the majority of the Vm-dependence is due to a Vm-dependent occupancy of ClC-2 pore by the permeant Cl- and that the open conformation is stabilized by a Vm-independent protonation and the Cl- occupancy. Supported by CONACyT.

Cardiac Ischemia and Ischemia/Reperfusion Cause Wide Proteolysis of the Coronary Endothelial Luminal Membrane: Possible Dysfunctions

Background:Ischemia and ischemia-reperfusion (I/R) are common clinical insults that disrupt the molecular structure of coronary vascular endothelial luminal membrane (VELM) that result in diverse microvasculature dysfunctions. However, the knowledge of the associated biochemical changes is meager. We hypothesized that ischemia and I/R-induced structural and functional VELM alterations result from biochemical changes. First, these changes need to be described and later the mechanisms behind be identified.Methods:During control conditions, in isolated perfused rat hearts VELM proteins were labeled with biotin. The groups of hearts were: control (C), no flow ischemia (I; 25 min), and I/R (I; 25 min, reperfusion 30 min). The biotinylated luminal endothelial membrane proteins in these three different groups were examined by 2-D electrophoresis and identified. But, it must be kept in mind the proteins were biotin-labeled during control.Results:A comparative analysis of the protein profiles under the 3 conditions following 2D gel electrophoresis showed differences in the molecular weight distribution such that MWC > MWI > MWI/R. Similar analysis for isoelectric points (pHi) showed a shift toward more acidic pHi under ischemic conditions. Of 100 % proteins identified during control 66% and 88% changed their MW-pHi during ischemia and I/R respectively. Among these lost proteins there were 9 proteins identified as adhesins and G-protein coupled receptors.General significance:I and I/R insults alter MW-pHi of most luminal glycocalyx proteins due to the activation of nonspecific hydrolizing mechanisms; suspect metalloproteases and glycanases. This makes necessary the identification of hydrolyzing enzymes reponsible of multiple microvascular dysfunctions in order to maintain the integrity of vascular endothelial membrane. VELM must become a target of future therapeutics.

The protective effect of brief acidic cardiac reperfusion after ischemia is not mimicked by inhibition of the Na + /H + exchanger NHE1 or of phospholipase A2‐VI (PLA2‐VI)

Postconditioning (PC) is a widely studied way to limit cardiac damage after ischemia, but the mechanisms involved are incompletely understood. Here, we investigated the effects of 15 min of acidic pH, or of inhibition of the Na+/H+ exchanger NHE1 or the Ca2+-independent phospholipase A2 (iPLA2-VI) at onset of reperfusion, on ischemia/reperfusion (I/R) damage in Langendorf-perfused rat hearts exposed to 35 min of global no-flow ischemia and 120 min reperfusion. Left ventricular end diastolic pressure (LVeDP) and infarct size were reduced and left ventricular developed pressure (LVDP) recovery improved to a comparable extent by PC (6 × 10 s I/R) or pH 6.0. In contrast, LVeDP and infarct size were increased and LVDP recovery attenuated by the NHE1 inhibitor EIPA and largely unaffected by the iPLA2-VI inhibitor BEL. In cultured HL-1 cardiomyocytes in presence of HCO3− (i.e. presumably capable of efficient pHi regulation) I/R elicited JNK-, Akt- and caspase-3 activation, PARP cleavage and necrotic cell death, neither of which were reduced by 15 min acidic PC or EIPA. In conclusion, PC or pH 6.0 at onset of reperfusion improved myocardial performance after I/R in whole hearts, whereas inhibition of NHE1 or iPLA2-VI were not protective. The protection of intact hearts by acidic pHi could not be mimicked in cultured HL-1 cardiomyocytes; the mechanisms underlying this are under investigation. Funding: Danish Heart Foundation; Danish National Research Council.

PH sensitivity of ammonium transport by Rhbg

Rhbg is a membrane glycoprotein that is involved in NH(3)/NH(4)(+) transport. Several models have been proposed to describe Rhbg, including an electroneutral NH(4)(+)/H(+) exchanger, a uniporter, an NH(4)(+) channel, or even a gas channel. In this study, we characterized the pH sensitivity of Rhbg expressed in Xenopus oocytes. We used two-electrode voltage clamp and ion-selective microelectrodes to measure NH(4)(+)-induced [and methyl ammonium (MA(+))] currents and changes in intracellular pH (pH(i)), respectively. In oocytes expressing Rhbg, 5 mM NH(4)Cl (NH(3)/NH(4)(+)) at extracellular pH (pH(o)) of 7.5 induced an inward current, decreased pH(i), and depolarized the cell. Raising pH(o) to 8.2 significantly enhanced the NH(4)(+)-induced current and pH(i) changes, whereas decreasing bath pH to 6.5 inhibited these changes. Lowering pH(i) (decreased by butyrate) also inhibited the NH(4)(+)-induced current and pH(i) decrease. In oocytes expressing Rhbg, 5 mM methyl amine hydrochloride (MA/MA(+)), often used as an NH(4)Cl substitute, induced an inward current, a pH(i) increase (not a decrease), and depolarization of the cell. Exposing the oocyte to MA/MA(+) at alkaline bath pH (8.2) enhanced the MA(+)-induced current, whereas lowering bath pH to 6.5 inhibited the MA(+) current completely. Exposing the oocyte to MA/MA(+) at low pH(i) abolished the MA(+)-induced current and depolarization; however, pH(i) still increased. These data indicate that 1) transport of NH(4)(+) and MA/MA(+) by Rhbg is pH sensitive; 2) electrogenic NH(4)(+) and MA(+) transport are stimulated by alkaline pH(o) but inhibited by acidic pH(i) or pH(o); and 3) electroneutral transport of MA by Rhbg is likely but is less sensitive to pH changes.

Na+-dependent HCO3− Import by the slc4a10 Gene Product Involves Cl− Export

The slc4a10 gene encodes an electroneutral Na(+)-dependent HCO(3)(-) importer for which the precise mode of action remains unsettled. To resolve this issue, intracellular pH (pH(i)) recordings were performed upon acidification in the presence of CO(2)/HCO(3)(-) by 2',7'-bis(carboxyethyl)-5,6-carboxyfluorescein (BCECF) fluorometry of stably slc4a10-transfected NIH-3T3 fibroblasts. slc4a10 expression induced a significant Na(+)-dependent pH(i) recovery, which was accompanied by an increase in the intracellular Na(+) concentration evaluated by use of the Na(+)-sensitive fluorophore CoroNa Green. The estimated Na(+):HCO(3)(-) stoichiometry was 1:2. Cl(-) is most likely the counterion maintaining electroneutrality because (i) Na(+)-dependent pH(i) recovery was eliminated in Cl(-)-depleted cells; (ii) acute extracellular Cl(-) removal led to a larger alkalization in slc4a10-transfected cells than in control cells; and (iii) the 4,4'-diisothiocyanato-stilbene-2,2'-disulfonic acid (DIDS)-sensitive and Na(+)- and HCO(3)(-)-dependent (36)Cl(-)-efflux during pH(i) recovery was significantly greater in acidified slc4a10-transfected cells than in control cells. Charged amino acids specific to slc4a gene family members that transport Na(+) and are expected to move more HCO(3)(-) molecules/turnover were targeted by site-directed mutagenesis. Na(+)-dependent pH(i) recovery was reduced in each of the single amino acid mutated cell lines (E890A, E892A, H976L, and H980G) compared with wild type slc4a10-transfected cells and completely eliminated in quadruple mutant cells. In conclusion, the data suggest that slc4a10 expressed in mammalian cells encodes a Na(+)-dependent Cl(-)/HCO(3)(-) exchanger in which four specific charged amino acids seem necessary for ion transport.

Intestinal Anion Exchanger Down-regulated in Adenoma (DRA) Is Inhibited by Intracellular Calcium

The Na/H exchanger 3 (NHE3) and the Cl/HCO(3) exchanger down-regulated in adenoma (DRA) together facilitate intestinal electroneutral NaCl absorption. Elevated Ca(2+)(i) inhibits NHE3 through mechanisms involving the PDZ domain proteins NHE3 kinase A regulatory protein (E3KARP) or PDZ kidney 1 (PDZK1). DRA also possesses a PDZ-binding motif, but the roles of interactions with E3KARP or PDZK1 and Ca(2+)(i) in DRA regulation are unknown. Wild type DRA and a mutant lacking the PDZ interaction motif (DRA-ETKFminus) were expressed constitutively in human embryonic kidney (HEK) and inducibly in Caco-2/BBE cells. DRA-mediated Cl/HCO(3) exchange was measured as intracellular pH changes. Ca(2+)(i) was assessed fluorometrically. DRA was induced 8-16-fold and was delivered to the apical surface of polarized Caco-2 cells. Putative anion transporter 1 and cystic fibrosis transmembrane regulator did not contribute to Cl/HCO(3) exchange in transfected Caco-2 cells. The calcium ionophore 4Br-A23187 inhibited DRA and DRA-ETKFminus in HEK cells, but only full-length DRA was inhibited in Caco-2 cells. In contrast, 100 microm UTP, which increased Ca(2+)(i), inhibited full-length DRA but not DRA-ETKFminus in Caco-2 and HEK cells. In HEK cells, which express little PDZK1, additional transfection of PDZK1 was required for UTP to inhibit DRA. As HEK cells do not express cystic fibrosis transmembrane regulator or NHE3, the data indicate that Ca(2+)(i)-dependent DRA inhibition is not because of modulation of other transport activities. In polarized epithelium, this inhibition requires interaction of DRA with PDZK1. Together with data from PDZK1(-/-) mice, these data underscore the prominent role of PDZK1 in Ca(2+)(i)-mediated inhibition of colonic NaCl absorption.

Identification of the large-conductance background K+ channel in mouse B cells as TREK-2

Mouse B cells and their cell line (WEHI-231) express large-conductance background K(+) channels (LK(bg)) that are activated by arachidonic acids, characteristics similar to TREK-2. However, there is no evidence to identify the molecular nature of LK(bg); some properties of LK(bg) were partly different from the reported results of TREK type channels. In this study, we compared the properties of cloned TREK-2 and LK(bg) in terms of their sensitivities to ATP, phosphatidylinositol 4,5-bisphosphate (PIP(2)), intracellular pH (pH(i)), and membrane stretch. Similar to the previous findings of LK(bg), TREK-2 showed spontaneous activation after membrane excision (i-o patch) and were inhibited by MgATP or by PIP(2). The inhibition by MgATP was prevented by wortmannin, suggesting membrane-delimited regulation of TREKs by phosphoinositide (PI) kinase. The same was observed with the property of LK(bg); the activation of TREK-2 by membrane stretch was suppressed by U73122 (PLC inhibitor). As with the known properties of TREK-2, LK(bg) were activated by acidic pH(i) and inhibited by PKC activator. Finally, we confirmed the expression of TREK-2 in WEHI-231 by using RT-PCR and immunoblot analyses. The amplitude of background K(+) current and the TREK-2 expression in WEHI-231 were commonly decreased by genetic knockdown of TREK-2 using small interfering RNA. The downregulation of TREK-2 attenuated Ca(2+)-influx induced by arachidonic acid in WEHI-231. As a whole, these results strongly indicate that TREK-2 encodes LK(bg) in mouse B cells. We also newly suggest that the low activity of TREK-2 in intact cells is due to the inhibition by intrinsic PIP(2).

Nonconserved charged residues of the AE2 transmembrane domain alter regulation of anion exchange by pH

We have investigated the molecular basis for regulation of the ubiquitous AE2/SLC4A2 anion exchanger by intracellular pH (pHi) and extracellular pH (pHo) through mutagenesis of C-terminal transmembrane domain (TMD) amino acid residues not conserved in the closely related but pH-insensitive AE1/SLC4A1. Selected conserved residues were also studied. AE2-mediated Cl−/Cl− exchange was measured as DIDS-sensitive 36Cl− efflux from Xenopus oocytes by varying pHi at constant pHo, and by varying pHo at near-constant pHi. Mutation of all 26 non-conserved charged residues of the AE2 TMD yielded functional protein, but mutations of 4 conserved charged residues reduced or abolished function. Wt pHi-sensitivity of anion exchange was reduced upon individual mutation of AE2 TMD residues R921, F922, P1077 and R1107, whereas TMD mutants K1153R, R1155K, R1202L displayed enhanced sensitivity to acidic pHi. In addition, individual mutagenesis of non-conserved AE2 TMD residues E981, K982 and D1075 to the corresponding AE1 residues significantly acid-shifted pHo-sensitivity. These results demonstrate that multiple conserved charged residues are important for basal transport function of AE2. They further suggests that multiple non-contiguous TMD amino acid residues contribute to regulation of AE2 by pH, and highlight the complexity of the molecular mechanisms by which pH regulates bicarbonate transporters.

Mechanism of Cardiac Sarcolemmal Adenosine Triphosphate–sensitive Potassium Channel Activation by Isoflurane in a Heterologous Expression System

Activation of the cardiac sarcolemmal adenosine triphosphate-sensitive potassium (KATP) channel during metabolic stress initiates cellular events that preserve cardiac performance. Previous studies showed that halogenated anesthetics prime KATP channels under whole cell voltage clamp and act in intracellular pH (pHi)-dependent manner on KATP channels in excised membrane patches. However, it is not known how halogenated anesthetics interact with these channels. The authors evaluated the effect of pHi and isoflurane on the KATP channel subunits, the pore-forming inward rectifier Kir6.2, and the regulatory sulfonylurea receptor SUR2A, using HEK293 cells as a heterologous expression system. Single channel activity was recorded in the inside-out patch configuration. At pHi 7.4, isoflurane had negligible effect on activity of wild-type Kir6.2/SUR2A, but at pHi 6.8, the channel open probability was increased by isoflurane (0.177 +/- 0.077 to 0.364 +/- 0.164). By contrast, the open probability of truncated Kir6.2DeltaC26, which forms a functional channel without SUR2A, was attenuated by isoflurane at both pHi 7.4 and pHi 6.8. Coexpression of Kir6.2DeltaC26 with SUR2A restored pHi sensitivity of channel activation by isoflurane. Site-directed mutagenesis within the Walker motifs of SUR2A abolished isoflurane activation of KATP channel at pHi 6.8. In addition, the pancreatic-type channels expressing sulfonylurea receptor SUR1 could not be activated by isoflurane. The nucleotide binding domains of SUR2A play a crucial role in isoflurane facilitation of the KATP channel activity at moderately acidic pHi as would occur during early ischemia. These findings support direct and differential interaction of isoflurane with the subunits of the cardiac sarcolemmal KATP channel.

Modulation of Kir4.2 rectification properties and pHi-sensitive run-down by association with Kir5.1

Inwardly rectifying K + channels (Kir) comprise seven subfamilies that can be subdivided further on the basis of cytosolic pH (pHi) sensitivity, rectification strength and kinetics, and resistance to run-down. Although distinct residues within each channel subunit define these properties, heteromeric association with other Kir subunits can modulate them. We identified such an effect in the wild-type forms of Kir4.2 and Kir5.1 and used this to further understand how the functional properties of Kir channels relate to their structures. Kir4.2 and a Kir4.2–Kir5.1 fusion protein were expressed in HEK293 cells. Inward currents from Kir4.2 were stable over 10 min and pHi-insensitive (pH 6 to 8). Conversely, currents from Kir4.2–Kir5.1 exhibited a pHi-sensitive run-down at slightly acidic pHi. At pHi 7.2, currents in response to voltage steps positive to E K were essentially time independent for Kir4.2 indicating rapid block by Mg 2+. Coexpression with Kir5.1 significantly increased the blocking time constant, and increased steady-state outward current characteristic of weak rectifiers. Recovery from blockade at negative potentials was voltage dependent and 2 to 10 times slower in the homomeric channel. These results show that Kir5.1 converts Kir4.2 from a strong to a weak rectifier, rendering it sensitive to pHi, and suggesting that Kir5.1 plays a role in fine-tuning Kir4.2 activity.

Activation-coupled inactivation in the bacterial potassium channel KcsA

X-ray structures of the bacterial K+ channel KcsA have led to unparalleled progress in our understanding of ion channel structures. The KcsA channel has therefore been a prototypic model used to study the structural basis of ion channel function, including the gating mechanism. This channel was previously found to close at near-neutral intracellular pH (pH(i)) and to open at acidic pH(i). Here, we report the presence of a previously unknown channel inactivation process that occurs after the KcsA channel is activated. In our experiments, mammalian cells transfected with a codon-optimized synthetic gene encoding the KcsA protein expressed K+-selective channels that activated in response to a decrease in pH(i). Using patch-clamp and rapid solution exchange techniques, we observed that the KcsA channels inactivated within hundreds of milliseconds after channel activation. At all tested pHs, inactivation always accompanied activation, and it was profoundly accelerated in the same pH range at which activation increased steeply. Recovery from inactivation was observed, and its extent depended on the pH(i) and the amount of time that the channel was inactive. KcsA channel inactivation can be described by a kinetic model in which pH(i) controls inactivation through pH-dependent activation. This heretofore-undocumented inactivation process increases the complexity of KcsA channel function, but it also offers a potential model for studying the structural correspondence of ion channel inactivation.