Low Internal pH Research Articles

Interaction between autophagic vesicles and the Coxiella-containing vacuole requires CLTC (clathrin heavy chain)

ABSTRACTCoxiella burnetii is an intracellular bacterial pathogen which causes Q fever, a human infection with the ability to cause chronic disease with potentially life-threatening outcomes. In humans, Coxiella infects alveolar macrophages where it replicates to high numbers in a unique, pathogen-directed lysosome-derived vacuole. This compartment, termed the Coxiella-containing vacuole (CCV), has a low internal pH and contains markers both of lysosomes and autophagosomes. The CCV membrane is also enriched with CLTC (clathrin heavy chain) and this contributes to the success of the CCV. Here, we describe a role for CLTC, a scaffolding protein of clathrin-coated vesicles, in facilitating the fusion of autophagosomes with the CCV. During gene silencing of CLTC, CCVs are unable to fuse with each other, a phenotype also seen when silencing genes involved in macroautophagy/autophagy. MAP1LC3B/LC3B, which is normally observed inside the CCV, is excluded from CCVs in the absence of CLTC. Additionally, this study demonstrates that autophagosome fusion contributes to CCV size as cell starvation and subsequent autophagy induction leads to further CCV expansion. This is CLTC dependent, as the absence of CLTC renders autophagosomes no longer able to contribute to the expansion of the CCV. This investigation provides a functional link between CLTC and autophagy in the context of Coxiella infection and highlights the CCV as an important tool to explore the interactions between these vesicular trafficking pathways.

Phosphate modified calcium aluminate cement for radioactive waste encapsulation

AbrstractThe present study is part of a wider investigation to develop an alternative cementing system for the encapsulation of problematic low and intermediate level radioactive waste. It has been suggested that alternative cementing systems, with lower internal pH than conventional Portland cement based composite cements, may reduce the corrosion of some reactive metals and may be beneficial for the long term durability of wasteforms. A potential alternative is an acid–base cementing system, based on mixing calcium aluminate cement (CAC) with acidic phosphate solutions. Although these systems have been studied previously, there has been no systematic investigation to identify phosphates for producing suitable matrixes for application in radioactive waste encapsulation. In the current study, monophosphate modified CAC formulations did not set or develop significant strength, whereas polyphosphate modified CAC formulations exhibited rapid setting and strength development. It is proposed that polyphosphate modified systems form amorphous reaction products, which act as binders between the partially and unreacted CAC particles, and were responsible for high strength development. Thermogravimetric analysis and scanning electron microscopy results suggest that this binding matrix consists of amorphous calcium phosphate and alumina gel. The results presented in this investigation suggest that polyphosphate modified CAC has potential as an alternative cementing system for radioactive waste encapsulation.

Saturable Uptake of Lipophilic Amine Drugs into Isolated Hepatocytes: Mechanisms and Consequences for Quantitative Clearance Prediction

The hepatic uptake of quinine, fluvoxamine, and fluoxetine (0.1-10 microM) was investigated with freshly isolated rat hepatocytes. The cell-to-medium concentration ratios (K(p)) were concentration-dependent: the mean maximum K(p) values (at 0.1 microM) were 150 (quinine), 500 (fluvoxamine), and 2000 (fluoxetine). There was also a large capacity site that was not saturable over the concentration range used (possibly partition into the phospholipid component of membranes); representing this site, the mean minimum K(p) values (at 10 microM) were 30 (quinine), 200 (fluvoxamine), and 500 (fluoxetine). To eliminate concomitant metabolism, cells were pretreated with the irreversible P450 inhibitor, aminobenzotriazole. The saturable uptake was substantially eliminated after exposure to carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (ATP inhibitor). The difference between the maximum and minimum K(p) for these three amine drugs, as well as for dextromethorphan, propranolol, and imipramine, was within a limited range of 3-fold, indicating a common magnitude of saturable uptake. Basic, permeable drugs are expected to be sequestered into lysosomes, which actively maintain their low internal pH (approximately 5) using ATP, and this process is predictable from the combined effects of pH-driven ion accumulation and unsaturable binding representing partition into membranes. The resultant predicted maximum K(p) correlated strongly with the observed maximum K(p). Thus, at low substrate concentrations, the fraction of drug unbound in the hepatocyte incubation (critical for assessing drug clearance and drug-drug interaction potential) may be dependent upon saturable as well as unsaturable binding, and for lipophilic, basic drugs, this can be readily estimated assuming a common degree of uptake into lysosomes.

Enhancing Macroautophagy Protects against Ischemia/Reperfusion Injury in Cardiac Myocytes

Cardiac myocytes undergo programmed cell death as a result of ischemia/reperfusion (I/R). One feature of I/R injury is the increased presence of autophagosomes. However, to date it is not known whether macroautophagy functions as a protective pathway, contributes to programmed cell death, or is an irrelevant event during cardiac I/R injury. We employed simulated I/R of cardiac HL-1 cells as an in vitro model of I/R injury to the heart. To assess macroautophagy, we quantified autophagosome generation and degradation (autophagic flux), as determined by steady-state levels of autophagosomes in relation to lysosomal inhibitor-mediated accumulation of autophagosomes. We found that I/R impaired both formation and downstream lysosomal degradation of autophagosomes. Overexpression of Beclin1 enhanced autophagic flux following I/R and significantly reduced activation of pro-apoptotic Bax, whereas RNA interference knockdown of Beclin1 increased Bax activation. Bcl-2 and Bcl-x(L) were protective against I/R injury, and expression of a Beclin1 Bcl-2/-x(L) binding domain mutant resulted in decreased autophagic flux and did not protect against I/R injury. Overexpression of Atg5, a component of the autophagosomal machinery downstream of Beclin1, did not affect cellular injury, whereas expression of a dominant negative mutant of Atg5 increased cellular injury. These results demonstrate that autophagic flux is impaired at the level of both induction and degradation and that enhancing autophagy constitutes a powerful and previously uncharacterized protective mechanism against I/R injury to the heart cell.

Subtype-specific, bi-component inhibition of SK channels by low internal pH

The effects of low intracellular pH (pH i 6.4) on cloned small-conductance Ca 2+-activated K + channel currents of all three subtypes (SK1, SK2, and SK3) were investigated in HEK293 cells using the patch-clamp technique. In 400 nM internal Ca 2+ [Ca 2+] i, all subtypes were inhibited by pH i 6.4 in the order of sensitivity: SK1 > SK3 > SK2. The inhibition increased with the transmembrane voltage. In saturating internal Ca 2+, the inhibition was abolished for SK1–3 channels at negative potentials, indicating a [Ca 2+] i-dependent mode of inhibition. Application of 50 μM 1-ethyl-2-benzimidazolone was able to potentiate SK3 current to the same extent as at neutral pH i. We conclude that SK1–3 all are inhibited by low pH i. We suggest two components of inhibition: a [Ca 2+] i-dependent component, likely involving the SK β-subunits calmodulin, and a voltage-dependent component, consistent with a pore-blocking effect. This pH i-dependent inhibition can be reversed pharmacologically.

Potassium-Dependent Slow Inactivation of Kir1.1 (ROMK) Channels

The ROMK (Kir1.1) family of epithelial K channels can be inactivated by a combination of low internal pH and low external K, such that alkalization does not reopen the channels unless external K is elevated. Previous work suggested that this inactivation results from an allosteric interaction between an inner pH gate and an outer K sensor, and could be described by a simple three-state kinetic model. In the present study, we report that a sustained depolarization slowly inactivated (half-time = 10–15 min) ROMK channels that had been engineered for increased affinity to internal polyamines. Furthermore, this inactivation occurred at external [K] ≤1 mM in ROMK mutants whose inner pH gate was constitutively open (ROMK2-K61M mutation). Both pH and voltage inactivation depended on external K in a manner reminiscent of C-type inactivation, but having a much slower time course. Replacement of ROMK extracellular loop residues by Kir2.1 homologous residues attenuated or abolished this inactivation. These results are consistent with the hypothesis that there are (at least) two separate closure processes in these channels: an inner pH-regulated gate, and an outer (inactivation) gate, where the latter is modulated by both voltage and external [K].

Comparison of unsteady- and steady-state methods for produce respiration rate determination

Equations were developed to predict the behavior of the measured respiration quotient (RQ measured ) during the measurement of produce respiration rates using various unsteady- and steady-state systems. These models predicted that the RQ measured determined in unsteady-state closed chambers would be a strong function of system loading and the produce’s internal pH whereas measurements in open steady-state systems would be independent of these variables. An examination of our previously published results with rutabaga ( Brassica napobrassica ) as well as other unsteady- and steady-state literature data obtained from various other produce verified the trends predicted by the models. The transient build-up of CO 2 species inside the produce under unsteady-state conditions, and the resulting metabolic changes that occur to compensate, can lead to large swings in RQ measured . In contrast, inside modified atmosphere packages (MAPs), when the produce is allowed to gradually reach steady-state conditions, RQ measured values remain relatively constant. It would appear that vegetables with a relatively high internal pH (e.g., lettuce or celery) are more sensitive to the presence of elevated levels of CO 2 whereas vegetables with lower internal pH (e.g., broccoli or spinach) or most of the fruits are less affected.

Introduction. Responses to weak acids, alkalinisation, biocides, UV irradiation, and toxic metal ions.

Polluting bacteria can be faced by a wide range of stressing agents and conditions in the aquatic environment, and the ability to resist these and survive depends on several factors (1). First, survival can be significantly affected by the inherent make-up of the organisms, some strains being inherently more stress resistant than others, even without stress exposure (2). Second, tolerance will be markedly influenced by the presence of inducible stress tolerance mechanisms (3,4). Third, the growth-phase will be influential, tolerance being especially substantial in the stationary-phase (1). Additionally, organisms will show greater stress tolerance if able to attach to surfaces (5), since attached organisms are frequently resistant to stress, while recent studies imply that filamentation may be a means of circumventing damaging stress effects (6,7). The articles here (8-11) will consider how certain stresses damage and kill bacteria, and some responses to these stresses. It should be noted at the outset that, frequently, organisms are exposed to more than one stress at the same time i.e. with inducible responses an organism may have to set-in-train more than one such response at the same time. Exposure to low pH values is a condition occurring in many situations (12). In the aquatic environment, effluents from chemical works and agricultural establishments can acidify natural waters, and industrial operations can also lead to acidification, following the precipitation of acid rain and acid snow (12). Foodstuffs are also commonly acidic, and there are many locations in the animal and human body where low pH exposures are the norm (13,14). In any of these locations, polluting, contaminating or infecting bacteria will be subjected to acid stress. In most of the above situations, the baleful effects of exposure to acidity are exacerbated by the presence of weak acids. Organisms able to resist external acidity due to inherent or induced tolerance, may not be able to resist the very low internal pH caused by the combination of low external pH plus weak acids, and may also be killed by weak acids alone (8). Several areas considered by Hirshfield et al. (8) are of great interest. The induction of antibiotic resistance (mar operon) by weak acids (15) is of major public health interest because such acids in the intestine could induce pathogens to resistance. In connection with this, it is useful that studies are now being made of envelope changes that increase or decrease inherent tolerance to weak acids. These complement the earlier studies of the role of outer membrane (OM) components (particularly the PhoE and OmpA porins (16,17)) in determining inherent sensitivity or tolerance to inorganic acid. It is surprising and disappointing that it appears (8), that there have been virtually no studies of the regulation of tolerance responses associated with weak acids, although there is some information for the mar operon, with a repressor and a transcriptional activator involved (15,18). Obviously there is a strong likelihood that extracellular sensing and induction components function here (see below), but this is only part of the story, as essentially no studies have been made of (1) what sensors may be involved, or (2) how they signal the switching-on of the responses etc., etc. There are probably studies already in the literature which might throw light on some aspects of how responses involving weak acids are controlled. For example, Roe et al. (19), showed that growth inhibition by acetate led to the accumulation of homocysteine and to methionine starvation. Such an effect will, in many E. coli strains, lead to a stringent response, with the increased production of (p)ppGpp. These nucleotides are known to play a role in many regulatory processes, so there is a possibility that they could be involved in the switching-on of some of the responses induced by weak acids. The natural environment, especially the aquatic environment, can suffer pollution by basic materials; these can enter from chemical and agricultural sources and from run-off from basic soils (12). …

Stimulatory Action of Internal Protons on Slo1 BK Channels

We investigated the internal pH-sensitivity of heterologously expressed hSlo1 BK channels. In the virtual absence of Ca(2+) and Mg(2+) to isolate the voltage-dependent gating transitions, low internal pH enhanced macroscopic hSlo1 currents by shifting the voltage-dependence of activation to more negative voltages. The activation time course was faster and the deactivation time course was slower with low pH. The estimated K(d) value of the stimulatory effect was approximately pH = 6.5 or 0.35 micro M. The stimulatory effect was maintained when the auxiliary subunit mouse beta1 was coexpressed. Treatment of the hSlo1 channel with the histidine modifying agent diethyl pyrocarbonate also enhanced the hSlo1 currents and greatly diminished the internal pH sensitivity, suggesting that diethyl pyrocarbonate and low pH may work on the same effector mechanism. High concentrations of Ca(2+) or Mg(2+) also masked the stimulatory effect of low internal pH. These results indicate that the acid-sensitivity of the Slo BK channel may involve the channel domain implicated in the divalent-dependent activation.

Interactive effects of low pH and high ammonium levels responsible for the decline of Cirsium dissectum (L.) Hill

The decline of Cirsium dissectum in dessicatingwetlands is attributed to acidification and eutrophication. Experimentalevidence was obtained for the first time on ammonium toxicity under low pH. Inahydroculture experiment, interactive effects of nitrogen forms (250μmol NH4 + or 250μmolNO3 −) and pH (4, 5 or 6) were studied with regardtothe vitality of C. dissectum seedlings. The results showthat 250 μmol l−1 ammonium as sole nitrogensource only had negative effects on C. dissectum incombination with a low pH. Ammonium uptake at a rhizosphere pH of 4, resultedinlower nitrogen contents of both roots and shoots, lower internal pH of rootsandshoots and increased contents of basic amino-acids, resulting indecreased survival rate and biomass development. At higher pH, or when nitratewas the nitrogen source, these processes do not take place. This phenomenonstresses the importance of periodic influence of base rich groundwater duringthe winter in wet species-rich heathlands and grasslands, necessary torestore the acid neutralising capacity of the soil. Anthropogenic lowering ofthe groundwater table will lead to acidification enabling ammonium to becometoxic to herbaceous plant species such as C. dissectum.

Susceptibility of the Invasive Seaweed Caulerpa taxifolia to Ionic Aluminium

The tropical green seaweed, Caulerpa taxifolia has been invading the northwestern Mediterranean Sea since 1984, and has been discovered recently in California. This strain of C. taxifolia has proved an aggressive colonist, capable of vigorous growth on a variety of substrates. It can displace the native seagrasses and seaweeds, and its progress remains unchecked by grazers, which either avoid it or are poisoned by it. A variety of unsuccessful attempts have been made to curtail its spread. In this report we document the susceptibility of C. taxifolia to ionic aluminium. The exposure of whole fronds to 1 mM AlCl3 causes complete inhibition of photosynthesis in less than 2 hours. The effect is irreversible. The ecologically important seagrass Posidonia oceanica is unaffected by this treatment. The unusually low internal pH of 4.0 found in Caulerpa taxifolia may explain its vulnerability to aluminium poisoning, since the highly toxic Al3+ ion predominates at low pH. The results of this study provide the basis for the development of an environmentally sound means of selectively reducing the dominance of the aggressive strain of C. taxifolia in areas where it adversely affects the native plants and animals.

PH-dependent Ca2+ binding to the F0 c-subunit affects proton translocation of the ATP synthase from Synechocystis 6803.

It was shown before (Wooten, D. C., and Dilley, R. A. (1993) J. Bioenerg. Biomembr. 25, 557-567; Zakharov, S. D., Li, X., Red'ko, T. P., and Dilley, R. A. (1996) J. Bioenerg. Biomembr. 28, 483-493) that pH dependent reversible Ca2+ binding near the N- and C-terminal end of the 8 kDa subunit c modulates ATP synthesis driven by an applied pH jump in chloroplast and E. coli ATP synthase due to closing a "proton gate" proposed to exist in the F0 H+ channel of the F0F1 ATP synthase. This mechanism has further been investigated with the use of membrane vesicles from mutants of the cyanobacterium Synechocystis 6803. Vesicles from a mutant with serine at position 37 in the hydrophilic loop of the c-subunit replaced by the charged glutamic acid (strain plc 37) has a higher H+/ATP ratio than the wild type and therefore shows ATP synthesis at low values of deltamuH+. The presence of 1 mM CaCl2 during the preparation and storage of these vesicles blocked acid-base jump ATP formation when the pH of the acid side (inside) was between pH 5.6 and 7.1, even though the deltapH of the acid-base jump was thermodynamically in excess of the necessary energy to drive ATP formation at an external pH above 8.28. That is, in the absence of added CaCl2, ATP formation did occur under those conditions. However, when the base stage pH was 7.16 and the acid stage below pH 5.2, ATP was formed when Ca2+ was present. This is consistent with Ca2+ being displaced by H+ ions from the F0 on the inside of the thylakoid membrane at pH values below about 5.5. Vesicles from a mutant with the serine of position 3 replaced by a cysteine apparently already contain some bound Ca2+ to F0. Addition of 1 mM EGTA during preparation and storage of those vesicles shifted the otherwise already low internal pH needed for onset of ATP synthesis to higher values when the external pH was above 8. With both strains it was shown that the Ca2+ binding effect on acid-base induced ATP synthesis occurs above an internal pH of about 5.5. These results were corroborated by 45Ca2+-ligand blot assays on organic solvent soluble preparations containing the 8 kDa F0 subunit c from the S-3-C mutant ATP synthase, which showed 5Ca2+ binding as occurs with the pea chloroplast subunit III. The phosphorylation efficiency (P/2e), at strong light intensity, of Ca2+ and EGTA treated vesicles from both strains were almost equal showing that Ca2+ or EGTA have no other effect on the ATP synthase such as a change in the proton to ATP ratio. The results indicate that the Ca2+ binding to the F0 H+ channel can block H+ flux through the channel at pH values above about 5.5, but below that pH protons apparently displace the bound Ca2+, opening the CF0 H+ channel between the thylakoid lumen and H+ conductive channel.

Histidine 118 in the S2–S3 Linker Specifically Controls Activation of the KAT1 Channel Expressed in Xenopus Oocytes

The guard cell K + channel KAT1, cloned from Arabidopsis thaliana, is activated by hyperpolarization and regulated by a variety of physiological factors. Low internal pH accelerated the activation kinetics of the KAT1 channel expressed in Xenopus oocytes with a pK of approximately 6, similar to guard cells in vivo. Mutations of histidine-118 located in the putative cytoplasmic linker between the S2 and S3 segments profoundly affected the gating behavior and pH dependence. At pH 7.2, substitution with a negatively charged amino acid (glutamate, aspartate) specifically slowed the activation time course, whereas that with a positively charged amino acid (lysine, arginine) accelerated. These mutations did not alter the channel’s deactivation time course or the gating behavior after the first opening. Introducing an uncharged amino acid (alanine, asparagine) at position 118 did not have any obvious effect on the activation kinetics at pH 7.2. The charged substitutions markedly decreased the sensitivity of the KAT1 channel to internal pH in the physiological range. We propose a linear kinetic scheme to account for the KAT1 activation time course at the voltages where the opening transitions dominate. Changes in one forward rate constant in the model adequately account for the effects of the mutations at position 118 in the S2–S3 linker segment. These results provide a molecular and biophysical basis for the diversity in the activation kinetics of inward rectifiers among different plant species.

Effect of several additives and their admixtures on the physico-chemical properties of a calcium phosphate cement.

Combinations of citrate (C6H5O(7)3-), pyrophosphate (P2O(7)4-) and sulfate (SO(4)2-) ions were used to modify the physico-chemical properties of a calcium phosphate cement (CPC) composed of beta-tricalcium phosphate (beta-TCP) and phosphoric acid (PA) solution. The results obtained with only one additive at a time are similar to those previously published. New facts are: the positive effect of C6H5O(7)3- ions on cement failure strain and their negative effect on cement pH. The position of the setting time maximum measured at an SO(4)2- concentration of 0.09 M was not displaced by the addition of C6H5O(7)3- and P2O(7)4- ions. However, the effect of SO(4)2- ions on the setting time was depressed by C6H5O(7)3- ions. Moreover, no increase in tensile strength was observed when increasing amounts of SO(4)2- were added into a C6H5O(7)3--containing cement. The latter results suggest a competitive effect of C6H5O(7)3- and SO(4)2- on setting time and tensile strength. Anhydrous dicalcium phosphate (DCP; CaHPO4) appeared in cement samples dried just after setting, but not in cement samples incubated for 24 h in deionized water before the drying step. It is believed that the setting reaction is stopped by the drying step, leaving a low internal pH in the sample, hence providing favorable conditions for the transformation of dicalcium phosphate dihydrate (DCPD) into DCP. Interestingly, even though C6H5O(7)3- ions dramatically lowered the equilibrium pH of the cement with 5 ml of deionized water, they still prevented the occurrence of the transformation of DCPD into DCP.

Rundown of the Hyperpolarization-Activated KAT1 Channel Involves Slowing of the Opening Transitions Regulated by Phosphorylation

Disappearance of the functional activity or rundown of ion channels upon patch excision in many cells involves a decrease in the number of channels available to open. A variety of cellular and biophysical mechanisms have been shown to be involved in the rundown of different ion channels. We examined the rundown process of the plant hyperpolarization-activated KAT1 K + channel expressed in Xenopus oocytes. The decrease in the KAT1 channel activity on patch excision was accompanied by progressive slowing of the activation time course, and it was caused by a shift in the voltage dependence of the channel without any change in the single-channel amplitude. The single-channel analysis showed that patch excision alters only the transitions leading up to the burst states of the channel. Patch cramming or concurrent application of protein kinase A (PKA) and ATP restored the channel activity. In contrast, nonspecific alkaline phosphatase (ALP) accelerated the rundown time course. Low internal pH, which inhibits ALP activity, slowed the KAT1 rundown time course. The results show that the opening transitions of the KAT1 channel are enhanced not only by hyperpolarization but also by PKA-mediated phosphorylation.

A 29 kDa Intracellular Chloride Channel p64H1 Is Associated with Large Dense-Core Vesicles in Rat Hippocampal Neurons

A novel class of intracellular chloride channels, the p64 family, has been found on several types of vesicles. These channels, acting in concert with the electrogenic proton pump, regulate the pH of the vesicle interior, which is critical for vesicular function. Here we describe the molecular cloning of p64H1, a p64 homolog, from both human and cow. Northern blot analysis showed that p64H1 is expressed abundantly in brain and retina, whereas the other members of this family (e.g., p64 and NCC27) are expressed only at low levels in these tissues. Immunohistochemical analysis of p64H1 in rat brain, using an affinity-purified antibody, revealed a high level of expression in the limbic system-the hippocampal formation, the amygdala, the hypothalamus, and the septum. Immunoelectron microscopic analysis of p64H1 in hippocampal neurons demonstrated a striking association between p64H1 and large dense-core vesicles (LDCVs) and microtubules. In contrast, very low p64H1 labeling was found in perikarya or associated with small synaptic vesicles (SSVs) in axonal profiles. Immunoblot analysis confirmed that p64H1 is colocalized with heavy membrane fractions containing LDCVs rather than the fractions containing SSVs. These results suggest that p64H1-mediated Cl- permeability may be involved in the maintenance of low internal pH in LDCVs and in the maturation of LDCVs and the biogenesis of functional neuropeptides.

Concentration and pH dependence of skeletal muscle chloride channel ClC-1.

1. The influence of Cl- concentration and pH on gating of the skeletal muscle Cl- channel, ClC-1, has been assessed using the voltage-clamp technique and the Sf-9 insect cell and Xenopus oocyte expression systems. 2. Hyperpolarization induces deactivating inward currents comprising a steady-state component and two exponentially decaying components, of which the faster is weakly voltage dependent and the slower strongly voltage dependent. 3. Open probability (Po) and kinetics depend on external but not internal Cl- concentration. 4. A point mutation, K585E, in human ClC-1, equivalent to a previously described mutation in the Torpedo electroplaque chloride channel, ClC-0, alters the I-V relationship and kinetics, but retains external Cl- dependence. 5. When external pH is reduced, the deactivating inward currents of ClC-1 are diminished without change in time constants while the steady-state component is enhanced. 6. In contrast, reduced internal pH slows deactivating current kinetics as its most immediately obvious action and the Po curve is shifted in the hyperpolarizing direction. Addition of internal benzoate at low internal pH counteracts both these effects. 7. A current activated by hyperpolarization can be revealed at an external pH of 5.5 in ClC-1, which in some ways resembles currents due to the slow gates of ClC-0. 8. Gating appears to be controlled by a Cl(-)-binding site accessible only from the exterior and, possibly, by modification of this site by external protonation. Intracellular hydroxyl ions strongly affect gating either allosterically or by direct binding and blocking of the pore, an action mimicked by intracellular benzoate.

Inhibition of Ca2+-dependent Cl? channels from secretory epithelial cells by low internal pH

The actions of intracellular pH (pHi) on Ca(2+)-dependent Cl- channels were studied in secretory epithelial cells derived from human colon carcinoma (T84) and in isolated rat parotid acinar cells. Channel currents were measured with the whole cell voltage clamp technique with pipette solutions of different pH. Ca(2+)-dependent Cl- channels were activated by superfusing ionomycin to increase the intracellular calcium concentration ([Ca2+]i) or by using pipette solutions with buffered Ca2+ levels. Large currents were activated in T84 and parotid cells by both methods with pHi levels of 7.3 or 8.3. Little or no Cl- channel current was activated with pHi at 6.4. We used on-cell patch clamp methods to investigate the actions of low pHi on single Cl- channel current amplitude in T84 cells. Lowering the pHi had little or no effect on the current amplitude of a 8 pS Cl- channel, but did reduce channel activity. These results suggest that cytosolic acidification may be able to modulate stimulus-secretion coupling in fluid-secreting epithelia by inhibiting the activation of Ca(2+)-activated Cl- channels.

I-type Lectins

Lysosomes are intracellular membrane-bound organelles that perform the final degradation of many cellular macromolecules. This is achieved by the action of a number of lysosomal enzymes (originally called “acid hydrolases” because of the low internal pH characteristic of lysosomes). These enzymes are synthesized in the endoplasmic reticulum (ER) on membrane-bound ribosomes and traverse the ER-Golgi pathway along with other newly synthesized proteins. At the terminal Golgi compartment (the trans-Golgi network or TGN), they are segregated from all other glycoproteins and selectively delivered to lysosomes. In most “higher” animal cells, this specialized trafficking is achieved primarily by a specific glycan marker that is recognized by certain receptors. This chapter describes the discovery and characterization of this glycan-mediated biological system, which relies on recognition of glycans containing mannose-6-phosphate (M6P) by “P-type” lectins. This was the first clear-cut example of a biological role for glycans on mammalian glycoproteins and the first demonstrated link between glycoprotein biosynthesis and human disease. The interesting history of its discovery is therefore described in some detail.

Effects of Internal pH on the Acetylcholine Transporter of Synaptic Vesicles

Uptake of acetylcholine (ACh) by synaptic vesicles isolated from the electric organ of Torpedo was induced with an artificially imposed proton gradient. The gradient was formed by hyposmotic lysis and resealing of vesicles in a low pH buffer to form vesicular ghosts followed by sudden elevation of the pH of the ghost suspension. [3H]ACh accumulated rapidly, the proton gradient collapsed spontaneously within 5 min as monitored by [14C]methylamine uptake, and the accumulated ACh leaked out of the ghosts after 5 min. Vesamicol blocked both uptake and efflux of the [3H]ACh, demonstrating that both processes are mediated by the ACh transporter. The protonophore nigericin also blocked uptake very potently. Specific uptake was titrated with variable concentrations of [3H]ACh. It exhibited Km and Vmax values of approximately 200-500 microM and 7-30 nmol [3H]ACh/mg at 5 min, respectively, which are values close to those commonly observed for ATP-dependent uptake by intact vesicles. Specific uptake by ghosts was titrated with variable internal pH and constant external pH. It exhibited maximal uptake between internal pH 4.5 and 5.5. The dependence was very steep and could be fit best by assuming that the active form of the transporter requires protonation of two internal sites of apparent pK value of 5.3 +/- 0.2. A similar result was obtained when the uptake was titrated with variable internal pH with a constant thermodynamic driving force maintained by keeping the external pH approximately 2.6 units higher. The origin of the transport inhibition that sets in at very low internal pH values is not clear.(ABSTRACT TRUNCATED AT 250 WORDS)