Changes In pHo Research Articles

TASK inhibition by mild acidosis increases Ca2+ oscillations to mediate pH sensing in rat carotid body chemoreceptor cells.

Severe levels of acidosis (pH < 6.8) have been shown to cause a sustained rise in cytosolic Ca2+ concentration in carotid body Type 1 (glomus) cells. To understand how physiologically relevant levels of acidosis regulate Ca2+ signaling in glomus cells, we studied the effects of small changes in extracellular pH (pHo) on the kinetics of Ca2+ oscillations. A decrease in pHo from 7.4 to 7.3 (designated mild) and 7.2 (designated moderate) acidosis produced significant increases in the frequency and amplitude of Ca2+ oscillations. These effects of acidosis on Ca2+ oscillations were not blocked by NS383 and amiloride [acid-sensing ion channel (ASIC) inhibitors]. Mild and moderate levels of acidosis, however, caused a small but significant inhibition of two-pore domain acid-sensing K+ channels (TASK) (TASK-1- and TASK-3-like channels) and depolarized the cell by 6-13 mV. Acidosis-induced increase in Ca2+ oscillations was inhibited by nifedipine (1 µM; L-type Cav inhibitor) and by TTA-P2 (20 µM; T-type Cav inhibitor). Mild inhibition of TASK activity by N-[(2,4-difluorophenyl)methyl]-2'-[[[2-(4methoxyphenyl)acetyl]amino]methyl][1,1'-biphenyl]-2-carboxamide (A1899) (0.3 µM) and 1-[1-[6-[[1,1'-biphenyl]-4-ylcarbonyl)-5,6,7,8-tetrahydropyrido[4,3-d]pyrimidine-4-yl]-4-piperidinyl]-1-butanon (PK-THPP) (0.1 µM) increased Ca2+ oscillation frequency to levels similar to those observed with mild-moderate acidosis. Mild acidosis (pHo 7.3) and mild hypoxia (∼5%O2) produced similar levels of changes in the kinetics of Ca2+ oscillations. Block of tetraethylammonium (TEA)-sensitive Kv channels did not affect acid-induced increase in Ca2+ oscillations. Our study shows that mild and moderate levels of acidosis increase the frequency and amplitude of Ca2+ oscillations primarily by inhibition of TASK without involving ASICs, and suggests a major role of TASK for signal transduction in response to a physiological change in pHo.

The Role of the Primary Cilium in Sensing Extracellular pH.

Biosensors on the membrane of the vascular endothelium are responsible for sensing mechanical and chemical signals in the blood. Transduction of these stimuli into intracellular signaling cascades regulate cellular processes including ion transport, gene expression, cell proliferation, and/or cell death. The primary cilium is a well-known biosensor of shear stress but its role in sensing extracellular pH change has never been examined. As a cellular extension into the immediate microenvironment, the cilium could be a prospective sensor for changes in pH and regulator of acid response in cells. We aim to test our hypothesis that the primary cilium plays the role of an acid sensor in cells using vascular endothelial and embryonic fibroblast cells as in vitro models. We measure changes in cellular pH using pH-sensitive 2′,7′-biscarboxyethy1-5,6-carboxyfluorescein acetoxy-methylester (BCECF) fluorescence and mitogen-activated protein kinase (MAPK) activity to quantify responses to both extracellular pH (pHo) and intracellular pH (pHi) changes. Our studies show that changes in pHo affect pHi in both wild-type and cilia-less Tg737 cells and that the kinetics of the pHi response are similar in both cells. Acidic pHo or pHi was observed to change the length of primary cilia in wild-type cells while the cilia in Tg737 remained absent. Vascular endothelial cells respond to acidic pH through activation of ERK1/2 and p38-mediated signaling pathways. The cilia-less Tg737 cells exhibit delayed responsiveness to pHo dependent and independent pHi acidification as depicted in the phosphorylation profile of ERK1/2 and p38. Otherwise, intracellular pH homeostatic response to acidic pHo is similar between wild-type and Tg737 cells, indicating that the primary cilia may not be the sole sensor for physiological pH changes. These endothelial cells respond to pH changes with a predominantly K+-dependent pHi recovery mechanism, regardless of ciliary presence or absence.

Exotic properties of a voltage-gated proton channel from the snail Helisoma trivolvis.

Voltage-gated proton channels, HV1, were first reported in Helix aspersa snail neurons. These H+ channels open very rapidly, two to three orders of magnitude faster than mammalian HV1. Here we identify an HV1 gene in the snail Helisoma trivolvis and verify protein level expression by Western blotting of H. trivolvis brain lysate. Expressed in mammalian cells, HtHV1 currents in most respects resemble those described in other snails, including rapid activation, 476 times faster than hHV1 (human) at pHo 7, between 50 and 90 mV. In contrast to most HV1, activation of HtHV1 is exponential, suggesting first-order kinetics. However, the large gating charge of ∼5.5 e0 suggests that HtHV1 functions as a dimer, evidently with highly cooperative gating. HtHV1 opening is exquisitely sensitive to pHo, whereas closing is nearly independent of pHo Zn2+ and Cd2+ inhibit HtHV1 currents in the micromolar range, slowing activation, shifting the proton conductance-voltage (gH-V) relationship to more positive potentials, and lowering the maximum conductance. This is consistent with HtHV1 possessing three of the four amino acids that coordinate Zn2+ in mammalian HV1. All known HV1 exhibit ΔpH-dependent gating that results in a 40-mV shift of the gH-V relationship for a unit change in either pHo or pHi This property is crucial for all the functions of HV1 in many species and numerous human cells. The HtHV1 channel exhibits normal or supernormal pHo dependence, but weak pHi dependence. Under favorable conditions, this might result in the HtHV1 channel conducting inward currents and perhaps mediating a proton action potential. The anomalous ΔpH-dependent gating of HtHV1 channels suggests a structural basis for this important property, which is further explored in this issue (Cherny et al. 2018. J. Gen. Physiol. https://doi.org/10.1085/jgp.201711968).

Allosteric coupling between proximal C-terminus and selectivity filter is facilitated by the movement of transmembrane segment 4 in TREK-2 channel.

TREK-2, a member of two-pore-domain potassium channel family, regulates cellular excitability in response to diverse stimuli. However, how such stimuli control channel function remains unclear. Here, by characterizing the responses of cytosolic proximal C-terminus deletant (ΔpCt) and transmembrane segment 4 (M4)-glycine hinge mutant (G312A) to 2-Aminoethoxydiphenyl borate (2-APB), an activator of TREK-2, we show that the transduction initiated from pCt domain is allosterically coupled with the conformation of selectivity filter (SF) via the movements of M4, without depending on the original status of SF. Moreover, ΔpCt and G312A also exhibited blunted responses to extracellular alkalization, a model to induce SF conformational transition. These results suggest that the coupling between pCt domain and SF is bidirectional, and M4 movements are involved in both processes. Further mechanistic exploration reveals that the function of Phe316, a residue close to the C-terminus of M4, is associated with such communications. However, unlike TREK-2, M4-hinge of TREK-1 only controls the transmission from pCt to SF, rather than SF conformational changes triggered by pHo changes. Together, our findings uncover the unique gating properties of TREK-2, and elucidate the mechanisms for how the extracellular and intracellular stimuli harness the pore gating allosterically.

Influence of light on the apoplastic ph in microwounded cells of Chara corallina

Microscopic wounding of plant cell walls by pathogens or by feeding insects triggers the defense responses, including a sharp rise in pH at the cell surface (pHo). Using internodal cells of Chara corallina Klein ex Willd., we show here that the elevated pHo in the area of cell wall microincision decreases in darkness and increases on illumination. These pHo changes occurred specifically in cell areas affected by microincision and were lacking in intact areas with active pHotosynthesis (acid zones). Localized illumination of a remote cell region located upstream the cytoplasmic flow at a 1.5-mm distance from the analyzed area also caused a transient increase in pHo in the area of microwounding but had no such effect in unwounded cell regions having weakly acidic pHo. Apparently, the increase in pHo after wounding is mediated by a metabolite released from illuminated chloroplasts, which is transported with the cytoplasmic flow for long distances. The transient pHo increase in the area of cell wall incision after illumination of a distant cell region coincided with a temporal increase in chlorophyll fluorescence F’. This implies the concurrent influence of the transported reductant (presumably NADH) on light emission of chloroplasts and on the H+ flow across the plasmalemma. We suppose that the alkalinization of cell surface in the area of microincision arises from H+ consumption in the apoplast in association with the transmembrane electron transport from cytoplasmic reducing equivalents to molecular oxygen.

Pathophysiologic Changes in Extracellular pH Modulate Parathyroid Calcium-Sensing Receptor Activity and Secretion via a Histidine-Independent Mechanism.

The calcium-sensing receptor (CaR) modulates renal calcium reabsorption and parathyroid hormone (PTH) secretion and is involved in the etiology of secondary hyperparathyroidism in CKD. Supraphysiologic changes in extracellular pH (pHo) modulate CaR responsiveness in HEK-293 (CaR-HEK) cells. Therefore, because acidosis and alkalosis are associated with altered PTH secretion in vivo, we examined whether pathophysiologic changes in pHo can significantly alter CaR responsiveness in both heterologous and endogenous expression systems and whether this affects PTH secretion. In both CaR-HEK and isolated bovine parathyroid cells, decreasing pHo from 7.4 to 7.2 rapidly inhibited CaR-induced intracellular calcium (Ca(2+)i) mobilization, whereas raising pHo to 7.6 potentiated responsiveness to extracellular calcium (Ca(2+)o). Similar pHo effects were observed for Ca(2+)o-induced extracellular signal-regulated kinase phosphorylation and actin polymerization and for L-Phe-induced Ca(2+)i mobilization. Intracellular pH was unaffected by acute 0.4-unit pHo changes, and the presence of physiologic albumin concentrations failed to attenuate the pHo-mediated effects. None of the individual point mutations created at histidine or cysteine residues in the extracellular domain of CaR attenuated pHo sensitivity. Finally, pathophysiologic pHo elevation reversibly suppressed PTH secretion from perifused human parathyroid cells, and acidosis transiently increased PTH secretion. Therefore, pathophysiologic pHo changes can modulate CaR responsiveness in HEK-293 and parathyroid cells independently of extracellular histidine residues. Specifically, pathophysiologic acidification inhibits CaR activity, thus permitting PTH secretion, whereas alkalinization potentiates CaR activity to suppress PTH secretion. These findings suggest that acid-base disturbances may affect the CaR-mediated control of parathyroid function and calcium metabolism in vivo.

The Effect of Extracellular pH Changes on Intracellular pH and Nitric Oxide Concentration in Endothelial and Smooth Muscle Cells from Rat Aorta

AimsIt has been known for more than a century that pH changes can alter vascular tone. However, there is no consensus about the effects of pH changes on vascular response. In this study, we investigated the effects of extracellular pH (pHo) changes on intracellular pH (pHi) and intracellular nitric oxide concentration ([NO]i) in freshly isolated endothelial cells and cross sections from rat aorta.Main MethodsThe HCl was used to reduce the pHo from 7.4 to 7.0 and from 7.4 to 6.5; the NaOH was used to increase the pHo from 7.4 to 8.0 and from 7.4 to 8.5. The fluorescent dyes 5-(and-6)-carboxy SNARF-1, acetoxymethyl ester, acetate (SNARF-1) and diaminofluorescein-FM diacetate (DAF-FM DA) were employed to measure the pHi and [NO]i, respectively. The fluorescence intensity was measured in freshly isolated endothelial cells by flow cytometry and in freshly obtained aorta cross sections by confocal microscopy.Key FindingsThe endothelial and vascular smooth muscle pHi was increased at pHo 8.5. The extracellular acidification did not change the endothelial pHi, but the smooth muscle pHi was reduced at pHo 7.0. At pHo 8.5 and pHo 6.5, the endothelial [NO]i was increased. Both extracellular alkalinization and acidification increased the vascular smooth muscle [NO]i.SignificanceNot all changes in pHo did result in pHi changes, but disruption of acid-base balance in both directions induced NO synthesis in the endothelium and/or vascular smooth muscle.

Further Characterization of the Electrogenicity and pH Sensitivity of the Human Organic Anion-Transporting Polypeptides OATP1B1 and OATP1B3

Organic anion-transporting polypeptides (OATPs) are involved in the liver uptake of many endogenous and xenobiotic compounds, such as bile acids and drugs, respectively. Using Xenopus laevis oocytes and Chinese hamster ovary (CHO) cells expressing rat Oatp1a1, human OATP1B1, or OATP1B3, the sensitivity of these transporters to extracellular/intracellular pH (pHo/pHi) and changes in plasma membrane potential (ΔΨ) was investigated. In X. laevis oocytes, nonspecific plasma membrane permeability increased only at pHo below 4.5. Above this value, both using oocytes and CHO cells, extracellular acidification affected differently the specific transport of taurocholic acid (TCA) and estradiol 17β-d-glucuronide (E(2)17βG) by Oatp1a1 (stimulation), OATP1B1 (inhibition), and OATP1B3 (stimulation). Changes in substrate uptake in the presence of valinomycin (K(+)-ionophore), carbonyl cyanide 3-chlorophenylhydrazone and nigericin (protonophores), and amiloride (Na(+)/H(+)-inhibitor) and cation replacement in the medium were studied with fluorescent probes for measuring substrate uptake (cholylglycyl amidofluorescein) and changes in pHi (SNARF-4F) and ΔΨ [DilC(1)(5)]. The results suggest that activity of these three carriers is sodium/potassium-independent and affected differently by changes in pHo and ΔΨ: Oatp1a1 was confirmed to be an electroneutral anion exchanger, whereas the function of both OATP1B1 and OATP1B3 was markedly affected by the magnitude of ΔΨ. Moreover, electrophysiological measurements revealed the existence of a net anion influx associated to OATP1B1/OATP1B3-mediated transport of TCA, E(2)17βG, and estrone-3-sulfate. Furthermore, a leakage of Na(+) through OATP1B1 and OATP1B3, which is not coupled to substrate transport, was found. In conclusion, these results suggest that OATP1B1 and OATP1B3 are electrogenic transporters whose activity may be strongly affected under circumstances of displacement of local pH.

Residues Important for Nitrate/Proton Coupling in Plant and Mammalian CLC Transporters

Members of the CLC gene family either function as chloride channels or as anion/proton exchangers. The plant AtClC-a uses the pH gradient across the vacuolar membrane to accumulate the nutrient NO(3)(-) in this organelle. When AtClC-a was expressed in Xenopus oocytes, it mediated NO(3)(-)/H(+) exchange and less efficiently mediated Cl(-)/H(+) exchange. Mutating the "gating glutamate" Glu-203 to alanine resulted in an uncoupled anion conductance that was larger for Cl(-) than NO(3)(-). Replacing the "proton glutamate" Glu-270 by alanine abolished currents. These could be restored by the uncoupling E203A mutation. Whereas mammalian endosomal ClC-4 and ClC-5 mediate stoichiometrically coupled 2Cl(-)/H(+) exchange, their NO(3)(-) transport is largely uncoupled from protons. By contrast, the AtClC-a-mediated NO(3)(-) accumulation in plant vacuoles requires tight NO(3)(-)/H(+) coupling. Comparison of AtClC-a and ClC-5 sequences identified a proline in AtClC-a that is replaced by serine in all mammalian CLC isoforms. When this proline was mutated to serine (P160S), Cl(-)/H(+) exchange of AtClC-a proceeded as efficiently as NO(3)(-)/H(+) exchange, suggesting a role of this residue in NO(3)(-)/H(+) exchange. Indeed, when the corresponding serine of ClC-5 was replaced by proline, this Cl(-)/H(+) exchanger gained efficient NO(3)(-)/H(+) coupling. When inserted into the model Torpedo chloride channel ClC-0, the equivalent mutation increased nitrate relative to chloride conductance. Hence, proline in the CLC pore signature sequence is important for NO(3)(-)/H(+) exchange and NO(3)(-) conductance both in plants and mammals. Gating and proton glutamates play similar roles in bacterial, plant, and mammalian CLC anion/proton exchangers.

Contributions of CO2, pHi, and pHo to the signaling pathway of chemosensitive neurons in the locus coeruleus

Central chemosensitive neurons increase their firing rate (FR) upon exposure to hypercapnic acidosis (HA: 15% CO2, pHo 7.0). Multiple signaling pathways have been proposed to account for this increase including changes in Ca2+, CO2, pHi, and pHo. To determine the relative contributions of ΔpHi, ΔpHo, and CO2 to an increased FR, we used the rapid diffusion of a weak acid technique to clamp pHi. A change in pHi is believed to be the major signal in the chemosensitive (CS) response due to strong correlations between the degree of intracellular acidification and the increase of FR in HA (P&lt;0.05). In fact, an intracellular acidification (0.24 ± 0.04 U) with no change in pHo or CO2 elicits an increase in FR of 1.4 ± 0.5 Hz. However, this FR response can still be elicited without any change in pHi (1.4 ± 0.1 Hz). Only when both pHi and pHo are clamped, does hypercapnia not result in an increase in FR (0.4 ± 0.3 Hz in 5% CO2 to 0.16 ± 0.1 Hz in 15% CO2). Thus, the increase in FR seen upon HA with pHi clamped is likely due to the change in pHo (0.45 U) during the HA exposure. Our data suggest that ΔpHi and ΔpHo can both lead to an increase in FR in response to HA, while CO2 alone does not appear to have a significant contribution. It appears that ΔpHi or ΔpHo saturates the FR response because ΔFR with HA is not different than ΔFR with ΔpHi only or ΔpHo only. These data strongly support the multiple factors model of chemosensitivity. [NIH grants RO1 HL56683 and F32 HL80877].

Determinants of Anion-Proton Coupling in Mammalian Endosomal CLC Proteins

Many proteins of the CLC gene family are Cl(-) channels, whereas others, like the bacterial ecClC-1 or mammalian ClC-4 and -5, mediate Cl(-)/H(+) exchange. Mutating a "gating glutamate" (Glu-224 in ClC-4 and Glu-211 in ClC-5) converted these exchangers into anion conductances, as did the neutralization of another, intracellular "proton glutamate" in ecClC-1. We show here that neutralizing the proton glutamate of ClC-4 (Glu-281) and ClC-5 (Glu-268), but not replacing it with aspartate, histidine, or tyrosine, rather abolished Cl(-) and H(+) transport. Surface expression was unchanged by these mutations. Uncoupled Cl(-) transport could be restored in the ClC-4(E281A) and ClC-5(E268A) proton glutamate mutations by additionally neutralizing the gating glutamates, suggesting that wild type proteins transport anions only when protons are supplied through a cytoplasmic H(+) donor. Each monomeric unit of the dimeric protein was found to be able to carry out Cl(-)/H(+) exchange independently from the transport activity of the neighboring subunit. NO(3)(-) or SCN(-) transport was partially uncoupled from H(+) countertransport but still depended on the proton glutamate. Inserting proton glutamates into CLC channels altered their gating but failed to convert them into Cl(-)/H(+) exchangers. Noise analysis indicated that ClC-5 switches between silent and transporting states with an apparent unitary conductance of 0.5 picosiemens. Our results are consistent with the idea that Cl(-)/H(+) exchange of the endosomal ClC-4 and -5 proteins relies on proton delivery from an intracellular titratable residue at position 268 (numbering of ClC-5) and that the strong rectification of currents arises from the voltage-dependent proton transfer from Glu-268 to Glu-211.

Relationships Between Calcium and pH in the Regulation of the Slow Afterhyperpolarization in Cultured Rat Hippocampal Neurons

The Ca(2+)-dependent slow afterhyperpolarization (AHP) is an important determinant of neuronal excitability. Although it is established that modest changes in extracellular pH (pH(o)) modulate the slow AHP, the relative contributions of changes in the priming Ca(2+) signal and intracellular pH (pH(i)) to this effect remain poorly defined. To gain a better understanding of the modulation of the slow AHP by changes in pH(o), we performed simultaneous recordings of intracellular free calcium concentration ([Ca(2+)](i)), pH(i), and the slow AHP in cultured rat hippocampal neurons coloaded with the Ca(2+)- and pH-sensitive fluorophores fura-2 and SNARF-5F, respectively, and whole cell patch-clamped using the perforated patch technique. Decreasing pH(o) from 7.2 to 6.5 lowered pH(i), reduced the magnitude of depolarization-evoked [Ca(2+)](i) transients, and inhibited the subsequent slow AHP; opposite effects were observed when pH(o) was increased from 7.2 to 7.5. Although decreases and increases in pH(i) (at a constant pH(o)) reduced and augmented, respectively, the slow AHP in the absence of marked changes in preceding [Ca(2+)](i) transients, the inhibition of the slow AHP by decreases in pH(o) was correlated with low pH(o)-dependent reductions in [Ca(2+)](i) transients rather than the decreases in pH(i) that accompanied the decreases in pH(o). In contrast, high pH(o)-induced increases in the slow AHP were correlated with the accompanying increases in pH(i) rather than high pH(o)-dependent increases in [Ca(2+)](i) transients. The results indicate that changes in pH(o) modulate the slow AHP in a manner that depends on the direction of the pH(o) change and substantiate a role for changes in pH(i) in modulating the slow AHP during changes in pH(o).

Acidotic perfusion protects against ischemic injury by improving mitochondrial redox balance

Extracellular acidosis during ischemia reperfusion (IR) reduces cardiac injury due to slowed Na/H and Na/Ca exchange. We tested if acidosis also protects mitochondria. We compared effects of changes in pHo on altering redox state during IR. Guinea pig hearts were perfused with HEPES buffer to reduce changes in [Ca2+]o. Groups: pH 7.4, pH 7.0+16 mM K+ (cardioplegia, CP) and pH 8.0. After baseline (BL) hearts were perfused at one pH for 10 min before and 10 min after 35 min 37°C ischemia and then at pH 7.4 for 110 min. LVP and coronary flow (CF) were recorded and NADH, FAD and superoxide (O2·−) assessed by fluorescence (F) at the LV free wall. All data vs. BL. At 35 min ischemia O2·− F increased by 32±11, 79±3 and 84±5% in pH 7+CP, pH 7.4 and pH 8; NADH F decreased by 5±1, and 8±1% in pH 7.4 and pH 8, but increased in pH 7+CP by 11±2%; FAD F decreased in pH 7+CP and increased in pH 7.4 and pH 8. At 60 min reperfusion, LVP and CF (100% BL) were 30±8% and 51±5% (pH 7.4), 60±8% and 76±5% (pH 7+CP), and 18±4% and 20±6% (pH 8); NADH, FAD and O2·− F were less altered after pH 7+CP. This shows that low pH with CP, not only protects hearts, but also improves mitochondrial function during IR. Mitochondrial Ca2+ entry during IR is less at low pHo and higher at high pHo. Low pHo improved the redox state (less altered NADH and FAD), and reduced the rise in O2·− during ischemia, so the better return of contractility on reperfusion may be in part due to preserved mitochondrial function.

SLC26A7 Is a Cl– Channel Regulated by Intracellular pH

Members of the SLC26 transporter family play an essential role in several epithelial functions, as revealed by diseases associated with mutations in members of the family. Several members were shown to function as Cl(-) and HCO(3)(-) transporters that likely play an important role in epithelial Cl(-) absorption and HCO(3)(-) secretion. However, the mechanism of most transporters is not well understood. SLC26A7 is a member of the SLC26 transporter family reported to be expressed in the basolateral membrane of the cortical collecting duct and parietal cells and functions as a coupled Cl(-)/HCO(3)(-) exchanger. In the present work we examined the transport properties of SLC26A7 to determine its transport characteristics and electrogenicity. We found that when expressed in Xenopus oocytes or HEK293 cells SLC26A7 functions as a pH(i)-regulated Cl(-) channel with minimal OH(-)/HCO(3)(-) permeability. Expression of SLC26A7 in oocytes or HEK293 cells generated a Cl(-) current with linear I/V and an instantaneous current that was voltage- and time-independent. Based on measurement of reversal potential the selectivity of SLC26A7 is NO(3)(-)>>Cl(-)=Br(-)=I(-)>SO(4)(2-)=Glu(-), although I(-) partially inhibited the current. Incubating the cells with HCO(3)(-) or butyrate acidified the cytosol and increased the selectivity of SLC26A7 for Cl(-). Measurement of membrane potential and pH(i) showed minimal OH(-) and HCO(3)(-) transport by SLC26A7 when the cells were incubated in Cl(-)-containing or Cl(-)-free media. The activity of SLC26A7 was inhibited by all inhibitors of anion transporters tested, 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid, diphenylamine-2-carboxylic acid, and glybenclamide. These findings reveal that SLC26A7 functions as a unique Cl(-) channel that is regulated by intracellular H(+).

Acute pH-dependent Regulation of AE2-mediated Anion Exchange Involves Discrete Local Surfaces of the NH2-terminal Cytoplasmic Domain

We have previously defined in the NH2-terminal cytoplasmic domain of the mouse AE2/SLC4A2 anion exchanger a critical role for the highly conserved amino acids (aa) 336-347 in determining wild-type pH sensitivity of anion transport. We have now engineered hexa-Ala ((A)6) and individual amino acid substitutions to investigate the importance to pH-dependent regulation of AE2 activity of the larger surrounding region of aa 312-578. 4,4'-Diisothiocyanostilbene-2,2'-disulfonic acid (DIDS)-sensitive 36Cl- efflux from AE2-expressing Xenopus oocytes was monitored during changes in pHi or pHo in HEPES-buffered and in 5% CO2/HCO3- -buffered conditions. Wild-type AE2-mediated 36Cl- efflux was profoundly inhibited at low pHo, with a pHo(50) value = 6.75 +/- 0.05 and was stimulated up to 10-fold by intracellular alkalinization. Individual mutation of several amino acid residues at non-contiguous sites preceding or following the conserved sequence aa 336-347 attenuated pHi and/or pHo sensitivity of 36Cl- efflux. The largest attenuation of pH sensitivity occurred with the AE2 mutant (A)6357-362. This effect was phenocopied by AE2 H360E, suggesting a crucial role for His360. Homology modeling of the three-dimensional structure of the AE2 NH2-terminal cytoplasmic domain (based on the structure of the corresponding region of human AE1) predicts that those residues shown by mutagenesis to be functionally important define at least one localized surface region necessary for regulation of AE2 activity by pH.

Bis(2-ammonio-5-chloropyridine) tetradecachlorohexamolybdate

This thesis is based on a study of solid Molybdenum(II) chloride cluster compounds. The emphasis of the text is on the hydrogen network in the different compounds. The main analysis tool has been single crystal x-ray diffraction, and in some cases theoretical calculations have been performed to confirm the hydrogen positions.It has been shown that the Mo(II) cluster is a very good promoter for hydrogen bonds. The hydrogen bond networks studied here are fairly rigid and strong, except for one compound where the easy loss of a water molecule changes the water structure dramatically.There are several ways to alter the crystal structure and of course alter the hydrogen bond network.O Change the syntheses with respect to pHO Change the apical halogenidesO Introducing cations instead of H3O+O Using protonated organic amino bases as cations and different organic solventsAll of these methods have been tested and evaluated. The general conclusion is that the cluster units are nicely ordered in the hydrogen bond network.

Regulation of inwardly rectifying K+ channels in retinal pigment epithelial cells by intracellular pH.

Inwardly rectifying K+ (Kir) channels in the apical membrane of the retinal pigment epithelium (RPE) play a key role in the transport of K+ into and out of the subretinal space (SRS), a small extracellular compartment surrounding photoreceptor outer segments. Recent molecular and functional evidence indicates that these channels comprise Kir7.1 channel subunits. The purpose of this study was to determine whether Kir channels in the RPE are modulated by extracellular (pHo) or intracellular pH (pHi), both of which change upon illumination of the dark-adapted retina. The Kir current (IKir) in acutely dissociated bovine RPE cells was recorded in the whole-cell configuration while altering pHo or pHi. In cells dialysed with pipette solution buffered to pH 7.2, step changes in pHo from 7.4 to 8.0, 7.0 or 6.5 had little effect on IKir. Acidification to pHo 6.0, however, caused a transient activation of IKir followed by a slower inhibition. To determine the dependence of IKir on pHi, we altered pHi within individual RPE cells at constant pHo by imposing transmembrane acetate concentration gradients. These experiments revealed a biphasic relationship between IKir and pHi: IKir was maximal at about pHi 7.1, but decreased sharply at more acidic or alkaline levels. To evaluate the role of Kir7.1 channels in the pHi-dependent changes in IKir, we tested the effect of transmembrane acetate concentration gradients on Rb+ currents, which are 10-fold larger than K+ currents for this channel subtype. Inwardly rectifying Rb+ currents were maximal at about pHi 7.0 and were inhibited by intracellular alkalinization or acidification. We conclude that the Kir conductance in the RPE is modulated by intracellular pH in the physiological range and that this reflects the behaviour of Kir7.1 channels. This sensitivity to pHi may provide an important mechanism linking photoreceptor activity and RPE function.

Modulation of rat chorda tympani NaCl responses and intracellular Na+ activity in polarized taste receptor cells by pH.

Mixture interactions between sour and salt taste modalities were investigated in rats by direct measurement of intracellular pH (pHi) and Na+ activity ([Na+]i) in polarized fungiform taste receptor cells (TRCs) and by chorda tympani (CT) nerve recordings. Stimulating the lingual surface with NaCl solutions adjusted to pHs ranging between 2.0 and 10.3 increased the magnitude of NaCl CT responses linearly with increasing external pH (pHo). At pH 7.0, the epithelial sodium channel (ENaC) blocker, benzamil, decreased NaCl CT responses and inhibited further changes in CT responses induced by varying pHo to 2.0 or 10.3. At constant pHo, buffering NaCl solutions with potassium acetate/acetic acid (KA/AA) or HCO3 −/CO2 inhibited NaCl CT responses relative to CT responses obtained with NaCl solutions buffered with HEPES. The carbonic anhydrase blockers, MK-507 and MK-417, attenuated the inhibition of NaCl CT responses in HCO3 −/CO2 buffer, suggesting a regulatory role for pHi. In polarized TRCs step changes in apical pHo from 10.3 to 2.0 induced a linear decrease in pHi that remained within the physiological range (slope = 0.035; r2 = 0.98). At constant pHo, perfusing the apical membrane with Ringer's solutions buffered with KA/AA or HCO3 −/CO2 decreased resting TRC pHi, and MK-507 or MK-417 attenuated the decrease in pHi in TRCs perfused with HCO3 −/CO2 buffer. In parallel experiments, TRC [Na+]i decreased with (a) a decrease in apical pH, (b) exposing the apical membrane to amiloride or benzamil, (c) removal of apical Na+, and (d) acid loading the cells with NH4Cl or sodium acetate at constant pHo. Diethylpyrocarbonate and Zn2+, modification reagents for histidine residues in proteins, attenuated the CO2-induced inhibition of NaCl CT responses and the pHi-induced inhibition of apical Na+ influx in TRCs. We conclude that TRC pHi regulates Na+-influx through amiloride-sensitive apical ENaCs and hence modulates NaCl CT responses in acid/salt mixtures.

Quantification of the response of rat medullary raphe neurones to independent changes in pHo and PCO2

The medullary raphe nuclei contain putative central respiratory chemoreceptor neurones that are highly sensitive to acidosis. To define the primary stimulus for chemosensitivity in these neurones, the response to hypercapnic acidosis was quantified and compared with the response to independent changes in PCO2 and extracellular pH (pHo). Neurones from the ventromedial medulla of neonatal rats (P0-P2) were dissociated and maintained in tissue culture for long enough to develop a mature response (up to 70 days). Perforated patch clamp recordings were used to record membrane potential and firing rate while changes were made in pHo, PCO2 and/or [NaHCO3]o from baseline values of 7.4, 5 % and 26 mm, respectively. Hypercapnic acidosis (PCO2 9 %; pHo 7.17) induced an increase in firing rate to 285 % of control in one subset of neurones (‘stimulated neurones’) and induced a decrease in firing rate to 21 % of control in a different subset of neurones (‘inhibited neurones’). Isocapnic acidosis (pHo 7.16; [NaHCO3]o 15 mm) induced an increase in firing rate of stimulated neurones to 309 % of control, and a decrease in firing rate of inhibited neurones to 38 % of control. In a different group of neurones, isohydric hypercapnia (9 % PCO2; [NaHCO3]o 40 mm) induced an increase in firing rate of stimulated neurones by the same amount (to 384 % of control) as in response to hypercapnic acidosis (to 327 % of control). Inhibited neurones also responded to isohydric hypercapnia in the same way as they did to hypercapnic acidosis. In Hepes-buffered solution, both types of neurone responded to changes in pHo in the same way as they responded to changes in pHo in bicarbonate-buffered Ringer solution. It has previously been shown that all acidosis-stimulated neurones in the medullary raphe are immunoreactive for tryptophan hydroxylase (TpOH-ir). Here it was found that TpOH-ir neurones in the medullary raphe were immunoreactive for carbonic anhydrase type II and type IV (CA II and CA IV). However, CA immunoreactivity was also common in neurones of the hypoglossal motor nucleus, inferior olive, hippocampus and cerebellum, indicating that its presence is not uniquely associated with chemosensitive neurones. In addition, under the conditions used here, acetazolamide (100 μm) did not have a significant effect on the response to hypercapnic acidosis. We conclude that chemosensitivity of raphe neurones can occur independently of changes in pHo, PCO2 or bicarbonate. The results suggest that a change in intracellular pH (pHi) may be the primary stimulus for chemosensitivity in these putative central respiratory chemoreceptor neurones.

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.