Alkaline pHo Research Articles

The insulin receptor family and protein kinase B (Akt) are activated in the heart by alkaline pH and α1-adrenergic receptors.

Insulin and insulin-like growth factor stimulate protein synthesis and cardioprotection in the heart, acting through their receptors (INSRs, IGF1Rs) and signalling via protein kinase B (PKB, also known as Akt). Protein synthesis is increased in hearts perfused at alkaline pHo to the same extent as with insulin. Moreover, α1-adrenergic receptor (α1-AR) agonists (e.g. phenylephrine) increase protein synthesis in cardiomyocytes, activating PKB/Akt. In both cases, the mechanisms are not understood. Our aim was to determine if insulin receptor-related receptors (INSRRs, activated in kidney by alkaline pH) may account for the effects of alkaline pHo on cardiac protein synthesis, and establish if α1-ARs signal through the insulin receptor family. Alkaline pHo activated PKB/Akt signalling to the same degree as insulin in perfused adult male rat hearts. INSRRs were expressed in rat hearts and, by immunoblotting for phosphorylation (activation) of INSRRs/INSRs/IGF1Rs, we established that INSRRs, together with INSRs/IGF1Rs, are activated by alkaline pHo. The INSRR/INSR/IGF1R kinase inhibitor, linsitinib, prevented PKB/Akt activation by alkaline pHo, indicating that INSRRs/INSRs/IGF1Rs are required. Activation of PKB/Akt in cardiomyocytes by α1-AR agonists was also inhibited by linsitinib. Furthermore, linsitinib inhibited cardiomyocyte hypertrophy induced by α1-ARs in cultured cells, reduced the initial cardiac adaptation (24 h) to phenylephrine in vivo (assessed by echocardiography) and increased cardiac fibrosis over 4 days. We conclude that INSRRs are expressed in the heart and, together with INSRs/IGF1Rs, the insulin receptor family provide a potent system for promoting protein synthesis and cardioprotection. Moreover, this system is required for adaptive hypertrophy induced by α1-ARs.

Dual regulation of the native ClC-K2 chloride channel in the distal nephron by voltage and pH.

ClC-K2, a member of the ClC family of Cl(-) channels and transporters, forms the major basolateral Cl(-) conductance in distal nephron epithelial cells and therefore plays a central role in renal Cl(-) absorption. However, its regulation remains largely unknown because of the fact that recombinant ClC-K2 has not yet been studied at the single-channel level. In the present study, we investigate the effects of voltage, pH, Cl(-), and Ca(2+) on native ClC-K2 in the basolateral membrane of intercalated cells from the mouse connecting tubule. The ∼10-pS channel shows a steep voltage dependence such that channel activity increases with membrane depolarization. Intracellular pH (pHi) and extracellular pH (pHo) differentially modulate the voltage dependence curve: alkaline pHi flattens the curve by causing an increase in activity at negative voltages, whereas alkaline pHo shifts the curve toward negative voltages. In addition, pHi, pHo, and extracellular Ca(2+) strongly increase activity, mainly because of an increase in the number of active channels with a comparatively minor effect on channel open probability. Furthermore, voltage alters both the number of active channels and their open probability, whereas intracellular Cl(-) has little influence. We propose that changes in the number of active channels correspond to them entering or leaving an inactivated state, whereas modulation of open probability corresponds to common gating by these channels. We suggest that pH, through the combined effects of pHi and pHo on ClC-K2, might be a key regulator of NaCl absorption and Cl(-)/HCO3 (-) exchange in type B intercalated cells.

Extracellular Cl(-) regulates human SO4 (2-)/anion exchanger SLC26A1 by altering pH sensitivity of anion transport.

Genetic deficiency of the SLC26A1 anion exchanger in mice is known to be associated with hyposulfatemia and hyperoxaluria with nephrolithiasis, but many aspects of human SLC26A1 function remain to be explored. We report here the functional characterization of human SLC26A1, a 4,4'-diisothiocyanato-2,2'-stilbenedisulfonic acid (DIDS)-sensitive, electroneutral sodium-independent anion exchanger transporting sulfate, oxalate, bicarbonate, thiosulfate, and (with divergent properties) chloride. Human SLC26A1-mediated anion exchange differs from that of its rodent orthologs in its stimulation by alkaline pHo and inhibition by acidic pHo but not pHi and in its failure to transport glyoxylate. SLC26A1-mediated transport of sulfate and oxalate is highly dependent on allosteric activation by extracellular chloride or non-substrate anions. Extracellular chloride stimulates apparent V max of human SLC26A1-mediated sulfate uptake by conferring a 2-log decrease in sensitivity to inhibition by extracellular protons, without changing transporter affinity for extracellular sulfate. In contrast to SLC26A1-mediated sulfate transport, SLC26A1-associated chloride transport is activated by acid pHo, shows reduced sensitivity to DIDS, and exhibits cation dependence of its DIDS-insensitive component. Human SLC26A1 resembles SLC26 paralogs in its inhibition by phorbol ester activation of protein kinase C (PKC), which differs in its undiminished polypeptide abundance at or near the oocyte surface. Mutation of SLC26A1 residues corresponding to candidate anion binding site-associated residues in avian SLC26A5/prestin altered anion transport in patterns resembling those of prestin. However, rare SLC26A1 polymorphic variants from a patient with renal Fanconi Syndrome and from a patient with nephrolithiasis/calcinosis exhibited no loss-of-function phenotypes consistent with disease pathogenesis.

Molecular basis of pH sensitivity of Orai1/stim1 channels

Changes of intracellular and extracellular pH are involved in a variety of physiological and pathological processes, in which regulation of the Ca2+ release activated Ca2+ channel (ICRAC) by pH has been implicated. Ca2+ entry mediated by ICRAC as well as native ICRAC currents have been shown to be regulated by acidic or alkaline pH. However, the mechanism by which the CRAC channel is regulated by internal pH (pHi) and external pH (pHo) has remained elusive. By generating a series of mutations in the channel pore region, intracellular and extracellular loops, transmembrane domains, and N‐ and C‐termini, we unexpectedly identified that the mutant E190D lost sensitivity to both acidic and alkaline pHo, and the mutant H155F markedly diminished the response to acidic and alkaline pHi. Thus, it appears that E190 in the TM3 is the external pH sensor, and H155 in the intracellular loop is the internal pHi sensor of Orai1/Stim1 channels. Our results provide a novel mechanism that changes of external and internal pH modulate ICRAC activity by directly regulating channel gating of the pore‐forming subunit Orai through residues E190 and H155. Given that pH changes can influence a variety of physiological/pathological functions, Orai/Stim channels may be an important mediator for various physiological and pathological processes associated with acidosis and alkalinization.

Substitution of transmembrane domain Cys residues alters pHo-sensitive anion transport by AE2/SLC4A2 anion exchanger

AE2/SLC4A2 is the most widely expressed of the Na(+)-independent SLC4 Cl(-)/HCO3 (-) exchangers and is essential for postnatal survival, but its structure remains unknown. We have generated and expressed a mouse AE2 construct devoid of transmembrane domain cysteine (Cys) residues, mAE2Cys-less, to enhance the utility of Cys-substitution mutagenesis for structural and structure-function studies of mAE2. mAE2Cys-less expressed in Xenopus oocytes exhibited partial reduction of stilbene disulfonate-sensitive anion exchange activity. This activity was independent of the mAE2 N-terminal cytosolic domain and was accompanied by near-normal surface expression, without change in K 1/2 for extracellular Cl(-). mAE2Cys-less exhibited wildtype activation of anion exchange by hypertonicity and by NH4Cl, and wildtype inhibition of anion exchange by acidic intracellular pH (pHi) in the absence of NH4 (+). However, inhibition of anion exchange by extracellular pH (pHo) exhibited an alkaline shifted pHo(50) value of at least 0.6-0.7 pH units. Although SO4 (2-) transport by mAE2Cys-less resembled wildtype mAE2 in its stimulation by acidic pHo, the absence of transmembrane domain Cys residues abrogated activation of oxalate transport by acidic pHo. The contrasting enhancement of SO4 (2-) transport by alkaline pHo in the mAE1 anion translocation pathway mutant E699Q (Am J Physiol Cell Physiol 295: C302) was phenocopied by the corresponding mutant E1007Q in both AE2 and AE2Cys-less. However, the absence of transmembrane domain Cys residues exacerbated the reduced basal anion transport function exhibited by this and other missense substitutions at AE2 residue E1007. AE2Cys-less will be a valuable experimental tool for structure-function studies of the SLC4 gene family, but its utility for studies of AE2 regulation by extracellular pH must be evaluated in the context of its alkaline-shifted pHo sensitivity, resembling that of AE2 gastric parietal cell variant AE2c1.

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.

Separate Gating Mechanisms Mediate the Regulation of K2P Potassium Channel TASK-2 by Intra- and Extracellular pH

TASK-2 (KCNK5 or K(2P)5.1) is a background K(+) channel that is opened by extracellular alkalinization and plays a role in renal bicarbonate reabsorption and central chemoreception. Here, we demonstrate that in addition to its regulation by extracellular protons (pH(o)) TASK-2 is gated open by intracellular alkalinization. The following pieces of evidence suggest that the gating process controlled by intracellular pH (pH(i)) is independent from that under the command of pH(o). It was not possible to overcome closure by extracellular acidification by means of intracellular alkalinization. The mutant TASK-2-R224A that lacks sensitivity to pH(o) had normal pH(i)-dependent gating. Increasing extracellular K(+) concentration acid shifts pH(o) activity curve of TASK-2 yet did not affect pH(i) gating of TASK-2. pH(o) modulation of TASK-2 is voltage-dependent, whereas pH(i) gating was not altered by membrane potential. These results suggest that pH(o), which controls a selectivity filter external gate, and pH(i) act at different gating processes to open and close TASK-2 channels. We speculate that pH(i) regulates an inner gate. We demonstrate that neutralization of a lysine residue (Lys(245)) located at the C-terminal end of transmembrane domain 4 by mutation to alanine abolishes gating by pH(i). We postulate that this lysine acts as an intracellular pH sensor as its mutation to histidine acid-shifts the pH(i)-dependence curve of TASK-2 as expected from its lower pK(a). We conclude that intracellular pH, together with pH(o), is a critical determinant of TASK-2 activity and therefore of its physiological function.

NH3 Is Involved in the [formula omitted] Transport Induced by the Functional Expression of the Human Rh C Glycoprotein

Renal ammonium (NH3 + NH4+) transport is a key process for body acid-base balance. It is well known that several ionic transport systems allow NH4+ transmembrane translocation without high specificity NH4+, but it is still debated whether NH3, and more generally, gas, may be transported by transmembrane proteins. The human Rh glycoproteins have been proposed to mediate ammonium transport. Transport of NH4+ and/or NH3 by the epithelial Rh C glycoprotein (RhCG) may be of physiological importance in renal ammonium excretion because RhCG is mainly expressed in the distal nephron. However, RhCG function is not yet established. In the present study, we search for ammonium transport by RhCG. RhCG function was investigated by electrophysiological approaches in RhCG-expressing Xenopus laevis oocytes. In the submillimolar concentration range, NH4Cl exposure induced inward currents (IAM) in voltage-clamped RhCG-expressing cells, but not in control cells. At physiological extracellular pH (pHo) = 7.5, the amplitude of IAM increased with NH4Cl concentration and membrane hyperpolarization. The amplitude of IAM was independent of external Na+ or K+ concentrations but was enhanced by alkaline pHo and decreased by acid pHo. The apparent affinity of RhCG for NH4+ was affected by NH3 concentration and by changing pHo, whereas the apparent affinity for NH3 was unchanged by pHo, consistent with direct NH3 involvement in RhCG function. The enhancement of methylammonium-induced current by NH3 further supported this conclusion. Exposure to 500 microm NH4Cl induced a biphasic intracellular pH change in RhCG-expressing oocytes, consistent with both NH3 and NH4+ enhanced influx. Our results support the hypothesis of a specific role for RhCG in NH3 and NH4+ transport.

Modulation of the volume-sensitive K+ current in Ehrlich ascites tumour cells by pH.

The effects of extracellular and intracellular pH (pHo and pHi respectively) on the regulatory volume decrease (RVD) response and on the volume-sensitive K+ and Cl- currents (IK,vol and ICl,vol respectively) were studied in Ehrlich ascites tumour cells. Alkaline pHo accelerated and acidic pHo decelerated the RVD response significantly. Intra- and extracellular alkalinisation increased the amplitude of IK,vol whereas acidification had an inhibitory effect. The magnitude of ICl,vol was not affected by changes in pHi or pHo. A significant reduction in the activation time for IK,vol after hypotonic cell swelling was observed upon moderate intracellular alkalinisation (to pHi 7.9). A further increase in pHi to 8.4 resulted in the spontaneous activation of an IK under isotonic conditions which resembled IK,vol with respect to its pharmacological profile and current/voltage (I/V) relation. Noise analysis demonstrated that the increased amplitude of IK,vol at alkaline pH resulted mainly from an increase in the number of channels (N) contributing to the current. The channel open probability, Po, was largely unaffected by pH. The pH dependence and the biophysical and pharmacological properties of IK,vol are similar to those of the cloned tandem pore-domain acid-sensitive K+ (TASK) channels, and in the current study the presence of TASK-1 was confirmed in Ehrlich cells.

Activation of a Potassium Conductance by Extracellular Alkaline pH in Oocytes of Xenopus laevis

Electrophysiological properties of Xenopus oocytes exposed to alkaline extracellular pH (pHo) were investigated by measuring whole-cell currents using the two-electrode voltage-clamp method. Alkaline pHo (8.5 - 10.5) elicited an outward current in a pHo-dependent manner with a concomitant increase in the membrane conductance. This outward-current response was dependent on K+ because it was suppressed by a K+ channel blocker tetraethy lammonium+ (20 mM), and the reversal potential of the response was in good agreement with the Nernst equation for K+. The response was not affected by pretreatment of oocytes with the acetoxymethyl ester of bis-(o-aminophenoxy)-ethane-N,N,N’N’-tetraacetic acid (10 μM), a membrane-permeant intracellular Ca2+ chelator, but it was augmented by forskolin (0.4 μM), a stimulant of adenylate cyclase. The outward-current response originates in the oocyte but not in the surrounding follicle cells because the current could still be evoked when follicle cells were removed by collagenase or when gap junctions connecting the oocyte membrane and follicle cells were blocked by 1-octanol (1 mM). It is concluded that the outward current elicited by alkaline pHo in Xenopus oocytes is dependent on the activation of K+ channels via the cAMP pathway and that the outward current originates in the oocyte rather than the surrounding follicle cells.

Ionic mechanisms of spontaneous GABAergic events in rat hippocampal slices exposed to 4-aminopyridine.

Ionic mechanisms of spontaneous GABAergic events in rat hippocampal slices exposed to 4-aminopyridine. J. Neurophysiol. 78: 2582-2591, 1997. Ion-selective (H+ and K+) microelectrode techniques as well as conventional extra- and intracellular recordings were used to study the ionic mechanisms of propagating spontaneous GABAergic events (SGEs) in rat hippocampal slices exposed to 4-aminopyridine (4-AP, 50-100 mu M). All experiments were made in the presence of antagonists of ionotropic glutamate receptors [10 mu M 6-nitro-7-sulphamoylbenzoquinoxaline-2,3-dione (NBQX) and 40 mu M -2-amino-5-phosphonopentanoic acid (AP5)]. The SGEs were composed of a negative-going change in field potential with a temporally coincident increase (0.7 +/- 0.3 mM; mean +/- SE) in extracellular K+ ([K+]o) and an alkaline transient (0.01-0.08 units) in extracellular pH (pHo) in stratum radiatum of the area CA1. Simultaneous intracellular recordings showed a triphasic hyperpolarization-depolarization-late hyperpolarization response in pyramidal cells. Application of pentobarbital sodium (PB, 100 mu M) decreased the interval between SGEs from a mean value of 35 to approximately 20 s and shortened the period of refractoriness of stimulus-evoked propagating events. This was accompanied by an increase in the amplitude of the field potential response of the [K+]o and the pHo shifts and of the depolarizing phase of the pyramidal-cell response. The SGEs were completely blocked by the gamma-aminobutyric acid-A (GABAA) receptor antagonist, picrotoxin (PiTX; 100 mu M). The amplitudes of the negative-going field potential and of the depolarizing phase of the pyramidal-cell response as well as the ionic shifts associated with SGEs were strongly suppressed in the nominal absence of CO2/HCO-3. There was a five-fold increase in the interevent interval, and propagating SGEs could not be evoked by stimuli given at intervals shorter than approximately 2-3 min. Exposure to inhibitors of carbonic anhydrase, benzolamide (BA; 10 micro M) or ethoxyzolamide (EZA; 50 mu M) fully blocked the alkaline pHo transients and turned them into acid shifts. The poorly membrane-permeant BA had no discernible effect on the other components of the SGEs, but application of EZA had effects reminiscent to those of CO2/HCO-3-free medium. Addition of the GABAA receptor-permeant weak-acid anion, formate (20 mM) reestablished the SGEs that were first suppressed by exposure to the CO2/HCO-3-free medium. No SGEs were seen in the presence of a similar concentration of the GABAA receptor-impermeant anion propionate. Unlike the alkaline transients associated with HCO-3-driven SGEs, those supported by formate were not blocked by BA. The present data suggest that an inward current carried by bicarbonate is necessary for the generation of SGEs and that the GABAA receptor-mediated excitatory coupling among GABAergic interneurons is essentially dependent on the availability of intracellular bicarbonate.

Ca<sup>2+</sup> Entry in Isolated Rat Cortical Collecting Duct Is pH- and Voltage-Sensitive

We studied effects of extracellular pH (pH₀), voltage and Ca2+-channel blockers on the basal and swelling-induced increase in intracellular Ca2+ ([Ca2+]i) activity of isolated rat cortical collecting duct (CCD) segments. [Ca2+]i and membrane voltages (Vm) were measured using the fura-2 method and the slow-whole-cell patch-clamp technique, respectively. Fura-2 fluorescence ratio (FR) at a pHo of 7.4 was 1.04 ± 0.02 (n = 121), corresponding to a calculated [Ca2+]i of 97 ± 6 nM. Depolarization of Vm by changes in extracellular K+ reduced FR (n = 6), and hyperpolarization of Vm increased FR (n = 6). Lowering pHo to 6.0 decreased FR by 0.07 ± 0.01 (n = 20), elevation of pHo to 8.0 induced an increase in FR by 0.14 ± 0.01 (n = 67) exclusively via Ca2+ influx. Ca2+ influx showed an increased Mn2+ quench of fura-2 fluorescence with alkaline pHo and a decreased quench with acidic pHo. The alkaline pHo-induced increase in FR was completely abolished by Ni2+ (5 mM, n = 6) and La3+ (10 µM, n = 8) and partially by Gd3+ (10 µM, n = 7). Cell swelling induced by a decrease in extracellular osmolality by 130 mosm led to an increase in FR of 0.46 ± 0.05 (n = 20). The swelling-induced increase was reduced at a pHo of 6.0 to 0.27 ± 0.06 (n = 10) and enhanced at pHo of 8.0 to 0.57 ± 0.10 (n = 8). Ni2+ (n = 6) and Gd3+ (n = 7) had no, and La3+ (n = 8) only a partial inhibitory effect on the swelling-induced increase in FR. Ni2+ quenched fura-2 fluorescence during cell swelling. Verapamil diminished the basal and swelling-activated Ca2+ influx only at a concentration > 1 µM. Neither nifedipine (1 µM, n = 7) nor nimodipine (1 µM, n = 5) had any effect on the swelling-induced increase in FR. In rat CCD [Ca2+]i is sensitive to pHo and Vm. Basal and cell swelling-induced Ca2+ influx is apparently mediated via different pathways. L-type Ca2+ channels are most likely not responsible for this Ca2+ permeability.

The effects of external pH on calcium channel currents in bullfrog sympathetic neurons

We have investigated the effects of external pH (pHo) on whole-cell calcium channel currents in bullfrog sympathetic neurons. The peak inward current increased at alkaline pHo and decreased at acidic pHo. We used tail currents to distinguish effects of pHo on channel gating and permeation. There were large shifts in the voltage dependence of channel activation (approximately 40 mV between pHo and 9.0 and pHo 5.6), which could be explained by binding of H+ to surface charge according to Gouy-Chapman theory. To examine the effects of pHo on permeation, we measured tail currents at 0 mV, following steps to + 120 mV to maximally activate the channels. Unlike most previous studies, we found only a approximately 10% reduction in channel conductance from pHo 9.0 to pHo 6.4, despite a approximately 25 mV shift of channel activation. At lower pHo the channel conductance did decrease, which could be described by binding of H+ to a site with pKa = 5.1. In some cells, there was a separate slow decrease in conductance at low pHo, possibly because of changes in internal pH. These results suggest that changes in current at pHo > 6.4 result primarily from a shift in the voltage dependence of channel activation. A H(+)-binding site can explain a rapid decrease in channel conductance at lower pHo. The surface charge affecting gating has little effect on the local ion concentration near the pore, or on the channel conductance.

Modulation of the glutamate-evoked release of arachidonic acid from mouse cortical neurons: involvement of a pH-sensitive membrane phospholipase A2

Excitatory synaptic transmission is associated with changes in both extracellular and intracellular pH. Using mouse cortical neurons in primary cultures, we studied the sensitivity of glutamate-evoked release of 3H-arachidonic acid (3H-AA) to changes in extracellular pH (pHo) and related intracellular pH (pHi). As pHo was shifted from 7.2 to 7.8, the glutamate-evoked release of 3H-AA was enhanced by approximately threefold. The effect of alkaline pHo on the glutamate response was rapid, becoming significant within 2 min. 3H-AA release, evoked by both NMDA and kainate, was also enhanced by pHo alkalinization. NMDA- and kainate-induced increase in free intracellular Ca2+ was unaffected by changing pHo from 7.2 to 7.8, indicating that the receptor-induced Ca2+ influx is not responsible for the pHo sensitivity of the glutamate-evoked release of 3H-AA. Alkalinization of pHi obtained by incubating neurons in the presence of HCO3- or NH4 enhanced the glutamate-evoked release of 3H-AA, while pHi acidification obtained by blockade of Na+/H+ and Cl-/HCO3- exchangers decreased the glutamate response. Membrane-bound phospholipase A2 (mPLA2) activity was stimulated by Ca2+ in a pH-dependent manner, increasing its activity as pH was shifted from 7.2 to 7.8. This pH profile corresponds to the pH profile of the glutamate-, NMDA- and kainate-evoked release of 3H-AA. Taken together, these results indicate that the glutamate-evoked release of 3H-AA may be mediated by the pH-sensitive mPLA2. Since excitatory neurotransmission mediated by glutamate results in both pHo and pHi changes and since AA enhances glutamatergic neurotransmission at both pre- and postsynaptic levels, the data reported here reveals a possible molecular mechanism whereby glutamate can modulate its own signalling efficacy in a pH-dependent manner by regulating the release of AA.

The human erythrocyte anion transport protein, band 3. Characterization of exofacial alkaline titratable groups involved in anion binding/translocation.

Chloride self-exchange across the human erythrocyte membrane at alkaline extracellular pH (pHO) and constant neutral intracellular pH (pH(i)) can be described by an exofacial deprotonatable reciprocating anion binding site model. The conversion of the transport system from the neutral to the alkaline state is related to deprotonation of a positively charged ionic strength- and substrate-sensitive group. In the absence of substrate ions ([ClO] = 0) the group has a pK of approximately 9.4 at constant high ionic strength (equivalent to approximately 150 mM KCl) and a pK of approximately 8.7 at approximately zero ionic strength. The alkaline ping-pong system (examined at constant high ionic strength) demonstrates outward recruitment of the binding sites with an asymmetry factor of approximately 0.2, as compared with the inward recruitment of the transport system at neutral pHO with an asymmetry factor of approximately 10. The intrinsic half-saturation constant for chloride binding, with [Cli] = [Clo], increased from approximately 30 mM at neutral to approximately 110 mM at alkaline pHO. The maximal transport rate was a factor of approximately 1.7 higher at alkaline pHO. This increase explains the stimulation of anion transport, the "modifier hump," observed at alkaline pHO. The translocation of anions at alkaline pHO was inhibited by deprotonation of another substrate-sensitive group with an intrinsic pK of approximately 11.3. This group together with the group with a pK of approximately 9.4 appear to form the essential part of the exofacial anion binding site. The effect of extracellular iodide inhibition on chloride transport as a function of pHO could, moreover, be simulated if three extracellular iodide binding constants were included in the model: namely, a competitive intrinsic iodide binding constant of approximately 1 mM in the neutral state, a self-inhibitor binding constant of approximately 120 mM in the neutral state, and a competitive intrinsic binding constant of approximately 38 mM in the alkaline state.

Effects of external pH on ionic currents in smooth muscle cells from the basilar artery of the guinea pig.

pHo is an important determinant of vascular tone in cerebral blood vessels. We investigated the effects of changes in pHo on isolated smooth muscle cells from the basilar artery of the guinea pig. Single cells contracted rapidly in response to an elevation in pHo (constant CO2), and contraction was blocked by nifedipine, suggesting a role for dihydropyridine-sensitive Ca2+ channels. In whole-cell patch-clamp experiments, changes in pHo (pHo 5.7-8.1, pHi 7.2 with 10 mM HEPES) strongly affected the amplitude of the peak Ca2+ channel current (10 mM Ba2+, +15 mV, holding potential of -55 mV), with an apparent pK of 6.9. The current-voltage curves were minimally shifted, indicating no important effect of surface charge. To separate the slowly inactivating L-type Ca2+ channel current from the more rapidly inactivating B-type current, the decaying portions of inward currents from cells studied with repetitive 1-second pulses (+15 mV, holding potential of -55 mV) were fit to a two-component model. Titration curves for the L-type and B-type currents indicated maximum increases by factors of 3.65 and 1.28 at alkaline pHo and gave apparent pK values of 7.71 and 6.47 (Hill coefficient unity). The time constant of inactivation for the B-type current at +15 mV was little affected by pHo, whereas that for the L-type current increased somewhat with increasing pHo. Additional experiments showed no significant effect of pHo on holding current or on voltage-activated outward currents (pCai 7 with 11 mM EGTA). Our results provide additional evidence for participation of Ca2+ channels in regulating basal tone in cerebral smooth muscle and indicate that pHo regulates current through slowly inactivating, dihydropyridine-sensitive L-type Ca2+ channels.

Depletion of the inositol 1,4,5-trisphosphate-sensitive intracellular Ca2+ store in vascular endothelial cells activates the agonist-sensitive Ca(2+)-influx pathway.

Previous studies in non-excitable cells have suggested that depletion of internal Ca2+ stores activates Ca2+ influx from the extracellular space via a mechanism that does not require stimulation of phosphoinositide hydrolysis. To test this hypothesis in vascular endothelial cells, the effect of the Ca(2+)-ATPase/pump inhibitor 2,5-di-t-butylhydroquinone (BHQ) on cytosolic free Ca2+ concentration ([Ca2+]i) was examined. BHQ produced a dose-dependent increase in [Ca2+]i, which remained elevated over basal values for several minutes and was substantially inhibited in the absence of extracellular Ca2+. Application of bradykinin after BHQ demonstrated that the BHQ-sensitive compartment partially overlapped the bradykinin-sensitive store. Similar results were obtained with thapsigargin and cyclopiazonic acid, two other Ca(2+)-ATPase inhibitors. Although BHQ had no effect on phosphoinositide hydrolysis, both 45Ca2+ influx and efflux were stimulated by this agent. These results suggest that depletion of the agonist-sensitive Ca2+ store is sufficient for activation of Ca2+ influx. Several characteristics of the Ca(2+)-influx pathway activated by internal store depletion were compared with those of the agonist-activated pathway. Bradykinin-stimulated Ca2+ influx was increased at alkaline extracellular pH (pHo), and was inhibited by extracellular La3+, by depolarization of the membrane, and by the novel Ca(2+)-influx blocker 1-(beta-[3-(4-methoxyphenyl)propoxy]-4- methoxyphenethyl)-1H-imidazole hydrochloride (SKF 96365). Additionally, bradykinin stimulated influx of both 45Ca2+ and 133Ba2+, consistent with the hypothesis that the agonist-activated influx pathway is permeable to both of these bivalent cations. Likewise, activation of Ca2+ influx by BHQ, thapsigargin and cyclopiazonic acid was blocked by La3+, membrane depolarization and SKF 96365, but was unaffected by nitrendipine or BAY K 8644. Furthermore, Ca2+ influx stimulated by BHQ was increased at alkaline pHo and BHQ stimulated the influx of both 45Ca2+ and 133Ba2+ to the same extent. These results demonstrate that the agonist-activated Ca(2+)-influx pathway and the pathway activated by depletion of the agonist-sensitive internal Ca2+ store are indistinguishable.

The cyanobacterium Synechococcus R‐2 (Anacystis nidulans, S. leopoliensis) PCC 7942 has a sodium‐dependent chloride transporter

ABSTRACT Synechococcus R‐2 (PCC 7942) actively accumulated Cl− in the light and dark, under control conditions (BG‐11 media: pHo, 7·5; [Na+]o, 18 mol m−3; [Cl−]o, 0·508 molm−3). In BG‐11 medium [Cl−], was 17·2±0·848 mol m−3 (light), electrochemical potential of Cl− (ΔμCl−i,o) =+211±2mV; [Cl−]i= 1·24±0·11 mol m−3(dark), ΔμCl−i,o=+133±4mV. Cl− fluxes, but not permeabilities, were much higher in the light: ϕCl−i,o= 4·01±5·4 nmol m−2 s−1, PCl−i,o= 47±5pm s−1 (light); ϕCl−i,o= 0·395±0·071 nmol m−2 s−1, PCl−i,o= 69±14 pm s−1 (dark). Chloride fluxes are inhibited by acid pHo (pHo 5; ϕCl−i,o= 0·14±0·04 nmol m−2 s−1); optimal at pHo 7·5 and not strongly inhibited by alkaline pHo (pHo 10; ϕCl−1i,o= 1·7±0·14 nmol m−2 s−1). A Cl−in/2H+in coporter could not account for the accumulation of Cl− alkaline pHo. Permeability of Cl− is very low, below 100pm s−1 under all conditions used, and appears to be maximal at pHo 7·5 (50–70 pm s−1) and minimal in acid pHo (20pm s−1). DCCD (dicyclohexyl‐carbodiimide) inhibited ϕCl−i,o in the light about 75% and [Cl−]i fell to 2·2±0·26 (4) mol m−3. Valinomycin had no effect but monensin severely inhibited Cl− uptake ([Cl−]i= 1·02±0·32 mol m−3; ϕCl−i,o= 0·20±0·1 nmol m−2 s−1). Vanadate (200 mmol m−3) accelerated the Cl− flux (ϕCl−i,o= 5·28±0·64 nmol m−2 s−1) but slightly decreased accumulation of Cl− ([Cl−], = 13·9±1·3 mol m−3) in BG‐11 medium but had no significant effect in Na+‐free media. DCMU (dichlorophenyldimethylurea) did not reduce [Cl−], or ϕCl−i,o to that found in the dark ([Cl−]i= 8·41±0·76 mol m−3; ϕCl−i,o= 2·06±0·36 nmol m−2 s−1). Synechococcus also actively accumulated Cl− in Na+‐free media, [Cl−]i was lower but ΔΨi,o hyperpolarized in Na+‐free media and so the ΔμCl−i,o was little changed ([Cl−]i= 7·98±0·698 mol m−3; ΔμCl−i,o=+203±3 mV). Net Cl− uptake was stimulated by Na+; Li+ acted as a partial analogue for Na+. Synechococcus has a Na+ activated Cl− transporter which is probably a primary 2Cl−/ATP pump. The Cl− pump is voltage sensitive. ΔμCl−i,o is directly proportional to ΔΨi,o(P»0·01%): ΔμCl−i,o= ‐1·487 (±0·102) ×ΔΨi,o, r= ‐0·983, n= 31. The ΔμCl−i,o increased (more positive) as the Δμi,o became more negative. The ΔμCl−i,o has no known function, but might provide a driving force for the uptake of micronutrients.

Modulation by extracellular pH of bradykinin-evoked activation of Ca(2+)-activated K+ channels in endothelial cells

We used the patch-clamp technique to investigate, via the activation of Ca(2+)-activated potassium channels [K(Ca2+)channels], the effects of extracellular pH (pHo) on the bradykinin (BK)-stimulated rise in cytosolic Ca2+ concentration in bovine aortic endothelial cells (BAE). In cell-attached experiments, the external application of BK caused a transient activation of the K(Ca2+) channels. Increasing pHo from 7.3 to 9 maintained the channel activity, which was not inhibited by withdrawing the agonist. The channel-activation process could be blocked either by removing external Ca2+ or by depolarizing the cells with a high-K+ external solution. These results indicate that the Ca2+ influx triggered by BK contributes in maintaining the agonist-evoked response in high pHo. Changes in pHo produced a slight increase in the intracellular pH (pHi) measured fluorimetrically with the H+ indicator dye 2',7-bis(carboxyethyl)5(6')-carboxyfluorescein. However, increasing pHi by the external application of NH4Cl at physiological pHo caused a rapid decline and not an increase in the K(Ca2+) channel activity triggered by BK. In fura-2-loaded cells, alkaline pHo had no effect on the time course of the Ca2+ response to BK in external Ca(2+)-free conditions, suggesting that the Ca2+ extrusion process is not affected by pHo. Our results suggest that the BK-evoked Ca2+ influx, which is required to reload internal Ca2+ stores, is controlled by a mechanism depending on extracellular H+.

Oxalate Transport in Renal Tubular Cells From Normal and Stone-Forming Animals

To investigate the cellular mechanism(s) underlying kidney stone disease, we examined oxalate uptake in suspensions of renal cortical and papillary cells derived from control and stone-forming animals. In control animals, both cortical and papillary cells exhibited a time-dependent accumulation of oxalate. This uptake was mediated both by passive diffusion and by one or more transport processes sensitive to the anion transport inhibitor, DIDS. Oxalate uptake was also markedly sensitive to extracellular pH, showing increased uptake at acidic pH outside (pHo) (6.0), and reduced uptake at alkaline pHo (8.0). In renal tubular cells from stone-forming animals, oxalate uptake was markedly altered. Uptake was significantly reduced in cortical cells, whereas it was significantly stimulated in papillary cells from the same animals. Since the observed changes in oxalate handling occurred only in stone-forming animals, it is possible that alterations in renal cell oxalate transport contribute to calcium oxalate stone formation.