The transport of chloride in Ehrlich ascites tumor cells.

The steady state transport and distribution of chloride between the intracellular and extracellular phases was investigated when the extracellular chloride concentration was varied by isosmotic replacement with nitrate, bromide and acetate. The results of these experiments show that chloride transport, measured by uptake of 36Cl, is sensitive to the replacement anion. In the presence of nitrate, chloride transport is a linear function of the extracellular chloride concentration. The relationship between chloride transport and extracellular chloride in the presence of bromide is concave upward which suggests that this anion inhibits chloride movement. However, when acetate replaces chloride, the relationship between chloride transport and extracellular chloride is concave downward. The chloride distribution ratio of cells incubated in 145-155mM chloride medium is 0.386 and is not effected by the replacement of chloride with nitrate, bromide or acetate. These findings are consistent with the assertion that chloride transport is composed of two parallel pathways, a diffusional plus a saturating, mediated component. Of the total chloride flux (9.1 mmoles Cl-/kg dry weight per minute) measured in chloride medium (145-155 mM Cl-), the mediated component represents 40% and the diffusional component 60%.     

Reversibly bound chloride in the atrial natriuretic peptide receptor hormone‐binding domain: Possible allosteric regulation and a conserved structural motif for the chloride‐binding site

The binding of atrial natriuretic peptide (ANP) to its receptor requires chloride, and it is chloride concentration dependent. The extracellular domain (ECD) of the ANP receptor (ANPR) contains a chloride near the ANP‐binding site, suggesting a possible regulatory role. The bound chloride, however, is completely buried in the polypeptide fold, and its functional role has remained unclear. Here, we have confirmed that chloride is necessary for ANP binding to the recombinant ECD or the full‐length ANPR expressed in CHO cells. ECD without chloride (ECD(?)) did not bind ANP. Its binding activity was fully restored by bromide or chloride addition. A new X‐ray structure of the bromide‐bound ECD is essentially identical to that of the chloride‐bound ECD. Furthermore, bromide atoms are localized at the same positions as chloride atoms both in the apo and in the ANP‐bound structures, indicating exchangeable and reversible halide binding. Far‐UV CD and thermal unfolding data show that ECD(?) largely retains the native structure. Sedimentation equilibrium in the absence of chloride shows that ECD(?) forms a strongly associated dimer, possibly preventing the structural rearrangement of the two monomers that is necessary for ANP binding. The primary and tertiary structures of the chloride‐binding site in ANPR are highly conserved among receptor‐guanylate cyclases and metabotropic glutamate receptors. The chloride‐dependent ANP binding, reversible chloride binding, and the highly conserved chloride‐binding site motif suggest a regulatory role for the receptor bound chloride. Chloride‐dependent regulation of ANPR may operate in the kidney, modulating ANP‐induced natriuresis.     

Chloride binding regulates the Schiff base pK in gecko P521 cone-type visual pigment

The binding of chloride is known to shift the absorption spectrum of most long-wavelength-absorbing cone-type visual pigments roughly 30 nm to the red. We determined that the chloride binding constant for this color shift in the gecko P521 visual pigment is 0.4 mM at pH 6.0. We found an additional effect of chloride on the P521 pigment: the apparent pKa of the Schiff base in P521 is greatly increased as the chloride concentration is increased. The apparent Schiff base pKa shifts from 8.4 for the chloride-free form to >10.4 for the chloride-bound form. We show that this shift is due to chloride binding to the pigment, not to the screening of the membrane surface charges by chloride ions. We also found that at high pH, the absorption maximum of the chloride-free pigment shifts from 495 to 475 nm. We suggest that the chloride-dependent shift of the apparent Schiff base pKa is due to the deprotonation of a residue in the chloride binding site with a pKa of ca. 8.5, roughly that of the Schiff base in the absence of chloride. The deprotonation of this site results in the formation of the 475 nm pigment and a 100-fold decrease in the pigment's ability to bind chloride. Increasing the concentration of chloride results in the stabilization of the protonated state of this residue in the chloride binding site and thus increased chloride binding with an accompanying increase in the Schiff base pK.     

On the relationship between anion binding and chloride conductance in the CFTR anion channel

Mutations at many sites within the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel pore region result in changes in chloride conductance. Although chloride binding in the pore – as well as interactions between concurrently bound chloride ions – are thought to be important facets of the chloride permeation mechanism, little is known about the relationship between anion binding and chloride conductance. The present work presents a comprehensive investigation of a number of anion binding properties in different pore mutants with differential effects on chloride conductance. When multiple pore mutants are compared, conductance appears best correlated with the ability of anions to bind to the pore when it is already occupied by chloride ions. In contrast, conductance was not correlated with biophysical measures of anion:anion interactions inside the pore. Although these findings suggest anion binding is required for high conductance, mutations that strengthened anion binding had very little effect on conductance, especially at high chloride concentrations, suggesting that the wild-type CFTR pore is already close to saturated with chloride ions. These results are used to support a revised model of chloride permeation in CFTR in which the overall chloride occupancy of multiple loosely-defined chloride binding sites results in high chloride conductance through the pore.     

Location of the binding site for chloride ion activation of cathepsin C.

Cathepsin C, a tetrameric lysosomal dipeptidyl-peptide hydrolase, is activated by chloride ion. The activation is shown here to be specific and pH-dependent, dissociation constants for chloride being lower at low pH. Bound chloride decreases the Km for the hydrolysis of chromophore labelled substrates without any significant change in Vmax, confirming its involvement in substrate binding. Determination of the kinetic parameters of chloride activation, using unlabelled substrates, has enabled its site of action to be located. The lower Km for the hydrolysis of simple amide substrates in the presence of Cl- shows that the S sites are involved. Possible involvement of the S' sites is excluded by the finding that the Km for the nucleophile in the transferase reaction is unaffected by chloride. The rates of inhibition by E-64 and iodoacetate are both chloride-dependent and, from the structure of the papain-E-64 complex, it is concluded that chloride binds close to the S2 site. The binding of guanidinium ion, a positively charged inhibitor, to the S site is dependent on chloride. Based on these results, a model is proposed to explain the chloride activation of cathepsin C. The possible physiological role of chloride in the regulation of proteolysis in the lysosome is discussed.     

The biology of simocephalus acutirostratus king (Cladocera: Daphnidae)-hatchability of the parthenogenetic egg cultured in artificial media

The development of the parthenogenetic egg of the daphnid, Simocephalus acutirostratus was studied in different artificial media. The hatchability is found to be 100% in 0.001 M to 0.01 M of sodium chloride. 100% hatchability is noticed in concentrations ranging from 0.001 M to 0.05 M of calcium chloride. Apart from minor fluctuations, a high percentage of hatchability is recorded when isotonic solutions of sodium and calcium chloride are mixed. The hatchability of the different stages of the embryo is studied in an artificial medium which contains various proportions of sodium chloride, potassium chloride and calcium chloride. The results are discussed and compared with those from an allied temperate form, Simocephalus vetulus (O. F. Muller).     

A9C sensitive Cl? - accumulation in A. thaliana root cells during salt stress is controlled by internal and external calcium

The involvement of chloride in salt stress symptoms and salt tolerance mechanisms in plants has been less investigated in the past. Therefore, we studied the salt-induced chloride influx in Arabidopsis expressing the GFP-based anion indicator Clomeleon. High salt concentrations induce two phases of chloride influx. The fast kinetic phase is likely caused by membrane depolarization, and is assumed to be mediated by channels. This is followed by a slower "saturation" phase, where chloride is accumulated in the cytoplasm. Both phases of chloride uptake are dependent on the presence of external calcium. In general: with high [Ca²⁺] less chloride is accumulated in the cytoplasm. Surprisingly, also the internal calcium availability has an impact on chloride transport. A complete block of the second phase of chloride influx is achieved by the anion channel blocker A9C and trivalent cations (La³⁺, Gd³⁺, and Al³⁺). Other channel blockers and diuretics were found to inhibit the process partially. The results suggest that several transporter species are involved here, including electroneutral cation-chloride-cotransporters, and a part of chloride possibly enters the cells through cation channels after salt application.     

Application of the general fluid circuit theory to the chloride-bromide-deuterium oxide experiment

The general fluid circuit theory of active chloride absorption is applied to an experiment in which chloride, bromide, and deuterium oxide were simultaneously absorbed from the lower ileum, chloride only moving against a concentration gradient. The conclusion is reached that the constant for active absorption of deuterium oxide is equal, within reasonable limits of error, to the constant for active chloride absorption in accordance with the assumption that the absorption of water carries chloride out of the intestinal lumen without changing its concentration.     

Properties of a halophil nicotinamide–adenine dinucleotide phosphate-specific isocitrate dehydrogenase. Preliminary studies of the salt relations and kinetics of the crude enzyme

The effects of chlorides on NADP-specific isocitrate dehydrogenase from Halobacterium salinarium were investigated. The enzyme is stabilized by potassium chloride and sodium chloride and this effect is discussed in relation to the Hill (1913) equation. Kinetics of the enzyme were studied within a range of concentrations of potassium chloride and sodium chloride. Apparent Michaelis constants for both substrates were affected by salt concentration, the effect being greater in sodium chloride than in potassium chloride. Minimal apparent Michaelis constants for both substrates were similar to the corresponding constants reported for yeast isocitrate dehydrogenase. V(max.) was maximal in each salt at a concentration of about 1m. The maximum was higher in sodium chloride than in potassium chloride. At salt concentrations above about 2.3m, the apparent V(max.) was lower in sodium chloride than in potassium chloride, and at salt concentrations below 0.75-1.0m, each salt behaved as a linear activator of the enzyme. Within this concentration range salt and NADP(+) acted competitively; the activation by salt was overcome at finite concentrations of NADP(+). At concentrations above about 1m, potassium chloride was a linear non-competitive inhibitor of the enzyme. Within the range 1.0-2.5m, sodium chloride was also a linear non-competitive inhibitor, but above 2.5m it caused more pronounced inhibition.     

Determination of chloride procedure based upon diffusion of hydrogen chloride

We describe a simple, inexpensive sample preparation method that involves the isolation of chloride as hydrogen chloride from serum and urine prior to chloride analysis with the chloride ion-selective electrode. Chloride analyses of clinical chemistry standards with the present method were found to be in good agreement with analyses reported by the manufacturer. Reliability of the method is also evident by complete recovery of chloride added to serum and urine, minimal day-to-day variation of analyses, and a coefficient of variation that generally is less than 2%. An evaluation of factors influencing the procedure is also reported. The usefulness of the chloride ion-selective electrode to determine chloride in serum or urine is greatly enhanced by the sample preparation method described since matrix interference by other sample components is removed prior to analysis.     

Chloride in smooth muscle

Interest in the functions of intracellular chloride expanded about twenty years ago but mostly this referred to tissues other than smooth muscle. On the other hand, accumulation of chloride above equilibrium seems to have been recognised more readily in smooth muscle.

Experimental data is used to show by calculation that the Donnan equilibrium cannot account for the chloride distribution in smooth muscle but it can in skeletal muscle. The evidence that chloride is normally above equilibrium in smooth muscle is discussed and comparisons are made with skeletal and cardiac muscle. The accent is on vascular smooth muscle and the mechanisms of accumulation and dissipation.

The three mechanisms by which chloride can be accumulated are described with some emphasis on calculating the driving forces, where this is possible. The mechanisms are chloride/bicarbonate exchange, (Na+K+Cl) cotransport and a novel entity, “pump III”, known only from own work. Their contributions to chloride accumulation vary and appear to be characteristic of individual smooth muscles. Thus, (Na+K+Cl) always drives chloride inwards, chloride/bicarbonate exchange is always present but does not always do it and “pump III” is not universal.

Three quite different biophysical approaches to assessing chloride permeability are considered and the calculations underlying them are worked out fully. Comparisons with other tissues are made to illustrate that low chloride permeability is a feature of smooth muscle.

Some of the functions of the high intracellular chloride concentrations are considered. This includes calculations to illustrate its depolarising influence on the membrane potential, a concept which, experience tells us, some people find confusing. The major topic is the role of chloride in the regulation of smooth muscle contractility. Whilst there is strong evidence that the opening of the calcium-dependent chloride channel leads to depolarisation, calcium entry and contraction in some smooth muscles, it appears that chloride serves a different function in others. Thus, although activation and inhibition of (Na+K+Cl) cotransport is associated with contraction and relaxation respectively, the converse association of inhibition and contraction has been seen. Nevertheless, inhibition of chloride/bicarbonate exchange and “pump III” and stimulation of (K+Cl) cotransport can all cause relaxation and this suggests that chloride is always involved in the contraction of smooth muscle.

The evidence that (Na+K+Cl) cotransport more active in experimental hypertension is discussed. This is a common but not universal observation. The information comes almost exclusively from work on cultured cells, usually from rat aorta. Nevertheless, work on smooth muscle freshly isolated from hypertensive rats confirms that (Na+K+Cl) cotransport is activated in hypertension but there are several other differences, of which the depolarisation of the membrane potential may be the most important.

Finally, a simple calculation is made which indicates as much as 40% of the energy put into the smooth muscle cell membrane by the sodium pump is necessary to drive (Na+K+Cl) cotransport. Notwithstanding the approximations in this calculation, this suggests that chloride accumulation is energetically expensive. Presumably, this is related to the apparently universal role of chloride in contraction.     

Typology of ephemerid chloride cells

Summary The tracheal gills of 16 species of mayfly larvae were studied with regard to the chloride cells. The ephemerid chloride cells occur as two main types: single cells and cell complexes. The single chloride cells are characterized by deep tubular or slit-like infoldings of the apical cell membrane, whereas the chloride cell complexes show numerous intercellular channels resulting from cellular interdigitation at the basolateral side. According to the structural organization of the apices, the ephemerid chloride cells may be classified into caviform, coniform, bulbiform and filiform types. In the caviform type (single chloride cell), the apex retracts to form an apical cavity similar to teleost chloride cells. In the other types (chloride cell complexes), there is a progressive extension of the central cell apex into or beyond the cuticle in the form of cones, bulbs or filaments. The common feature of all types is the differentiation of the cuticle into thin porous plates or envelopes covering or surrounding the various forms of apices.Histochemical precipitation of sodium and chloride in the apical region suggests that all types have basically the same function of salt absorption. The population of the various types differs with the species. However, there seem to be some taxonomic regularities with respect to the families. No relation was found between the types of chloride cells and habitat of the species.Supported by the National Science Foundation.     

Antiplatelet effects of chelerythrine chloride isolated from Zanthoxylum simulans

Chelerythrine chloride is an antiplatelet agent isolated from Zanthoxylum simulans. Aggregation and ATP release of washed rabbit platelets caused by ADP, arachidonic acid, PAF, collagen, ionophore A23187 and thrombin were inhibited by chelerythrine chloride. Less inhibition was observed in platelet-rich plasma. The thromboxane B2 formation of washed platelets caused by arachidonic acid, collagen, ionophore A23187 and thrombin was decreased by chelerythrine chloride. Phosphoinositides breakdown caused by collagen and PAF was completely inhibited by chelerythrine chloride, while that of thrombin was only partially suppressed. Chelerythrine chloride inhibited the intracellular calcium increase caused by arachidonic acid, PAF, collagen and thrombin in quin-2/AM-loaded platelets. The cyclic AMP level of washed platelets did not elevated by chelerythrine chloride. The antiplatelet effect of chelerythrine chloride was not dependent on the incubation time and the aggregability of platelets inhibited by chelerythrine chloride was easily recovered after sedimenting the platelets by centrifugation and then the platelet pellets were resuspended. Chelerythrine chloride did not cause any platelet lysis, since lactate dehydrogenase activity was not found in the supernatant. These data indicate that the inhibitory effect of chelerythrine chloride on rabbit platelet aggregation and release reaction is due to the inhibition on thromboxane formation and phosphoinositides breakdown.     

Relief from alkaline load in two-cell stage mouse embryos by bicarbonate/chloride exchange

Mouse embryos at the two-cell stage are able to recover from an alkaline load. We found that this recovery is mediated by sodium-independent bicarbonate/chloride exchange: intracellular pH (pHi) recovery from alkaline load is inhibited by the anion exchange inhibitor 4,4'-diisothiocyanostilbene disulfonic acid, lack of bicarbonate, or lack of chloride. The dependence of the pHi recovery on extracellular chloride concentration exhibits Michaelis-Menten kinetics. Furthermore, uptake of chloride is inhibited in a dose-dependent manner by extracellular bicarbonate. The Km for external chloride was found to be about 3 mM, with a Ki for external bicarbonate of about 2 mM. The exchanger is active above approximately pH 7.15. These results demonstrate that mouse embryos at the two-cell stage possess a sodium-independent bicarbonate/chloride exchange mechanism that is similar to that found in other mammalian cells. This bicarbonate/chloride exchanger appears to be the sole pHi-regulatory mechanism in the two-cell stage mouse embryo, since our previous results have shown that there are apparently no specific mechanisms active in these cells for relieving acid loads.     

A reexamination of the mechanisms underlying the arteriovenous chloride shift

The chloride shift is the movement of Cl(-) from the plasma into erythrocytes as blood moves from the arterial to the venous end of systemic capillaries. The traditional explanation for the chloride shift emphasizes the causative roles of the rise in Pco(2) and the exclusive presence of carbonic anhydrase within the red blood cell. The purpose of this article is, first, to reexamine two aspects of the chloride shift that we feel are traditionally underemphasized. They are the role of hemoglobin in causing the chloride shift and the affect of the chloride shift on the acid-base status of the blood. Second, we wish to reconcile more recent work with the traditional understanding of the chloride shift. The chloride shift has never been modeled from the perspective of the Stewart strong ion approach. Similarly, the traditional understanding has generally treated Cl(-) as a passive participant in the chloride shift whose role was simply to replace the lost negative charge of the outward moving HCO-3. More recent work has suggested that the ingoing Cl(-) is important for both O(2) unloading and acid-base balance of the blood. We conclude this article with a model of the chloride shift that uses the Stewart approach and, though harmonious with the traditional understanding, highlights the importance of hemoglobin and Cl(-) in the chloride shift.     

Nuclear Magnetic resonance quadrupole relaxation studies of chloride binding to the isolated hemoglobins from trout (Salmo irideus).

NMR studies of chloride binding to the main components of trout blood, Hb Trout I and Hb Trout IV, indicate that although the affinity of chloride is high for both hemoglobins, the characteristics of the binding process are markedly differnet. In Hb Trout IV chemical exchange at the chloride binding site(s) is fast and quadrupole effects determine the linewidth; chloride binding has a definite pH dependence, but there is no significant oxygen linkage. In contrast Hb Trout I represents a unique case of slow chemical exchange, which may depend on unusual stereoche mical characteristics of the chloride binding site; chloride binding is pH independent, but shows a significant oxygen linkage, which may be attributed to changes of the lifetime of chloride at the binding site. The chloride binding properties displayed by Hb Trout I and IV have been compared with those of normal and modified human hemoglobins and discussed in terms of the structural differences in the C- and N-terminal regions of the alpha- and beta-chains.     

Mechanism of a molecular valve in the halorhodopsin chloride pump

Halorhodopsin is a light-driven chloride anion pump in which the trans-->cis photoisomerization of a retinal chromophore triggers a photocycle resulting in the translocation of chloride across the plasma membrane. The mechanism of chloride transfer past the cis retinal is determined here by computing multiple pathways for this process. The calculations reveal two conditions of the valve mechanism. First, a lumen absent in the ground state structure is transiently opened by chloride passage. Second, this activated opening, which is achieved by flexible deformation of the surrounding protein, is shown to significantly raise the chloride translocation barrier between photocycles, thus preventing chloride backflow. Unlike macroscopic valve designs, the protein allows differential ion flows in the pumping and resting states that are tuned to match the physiological timescales of the cell, thus creating a "kinetic" valve.     

On the identity of the dissociation-linked chloride binding sites in human hemoglobin. Studies with hemoglobin modified with 4-isothiocyanatobenzenesulfonic acid and 4-isothiocyanatobenzenesulfonamide

The binding of chloride ions to specific sites on the human hemoglobin molecule has well-known effects on the oxygen equilibrium and on the stability of the tetrameric structure. Several lines of evidence suggest that the oxygen-linked and the dissociation-linked chloride binding sites differ. Direct evidence for this difference has been obtained from the chloride dependence of the dimer-tetramer equilibrium of oxyhemoglobin modified with 4-isothiocyanatobenzenesulfonic acid, in which all the oxygen-linked chloride binding sites are blocked, or with 4-isothiocyanatobenzenesulfonamide, in which the linkage between chloride and oxygen is unperturbed. Thus, the chloride dependence of the dimer-tetramer assembly is unaffected by the chemical modification in both proteins and resembles that of unreacted hemoglobin. It is suggested that histidines alpha-103, alpha-122 and beta-97 may constitute, at least in part, the dissociation-linked chloride binding sites.     

盐离子作用下组蛋白变体H2A.Z增强核小体结构的稳定性

真核生物染色质的基本结构组成单元是核小体,基因组DNA被压缩在染色质中,核小体的存在通常会抑制转录、复制、修复和重组等发生在DNA模板上的生物学过程。组蛋白变体H2A.Z可以调控染色质结构进而影响基因的转录过程,但其详细的调控机制仍未研究清楚。为了比较含有组蛋白变体H2A.Z的核小体和常规核小体在盐离子作用下的稳定性差异,本文采用Förster共振能量转移的方法检测氯化钠、氯化钾、氯化锰、氯化钙、氯化镁等离子对核小体的解聚影响。实验对Widom 601 DNA序列进行双荧光Cy3和Cy5标记,通过荧光信号值的变化来反映核小体的解聚变化。Förster共振能量转移检测结果显示:在氯化钠、氯化钾、氯化锰、氯化钙和氯化镁作用下,含有组蛋白变体H2A.Z的核小体解聚速度相比于常规核小体要慢,且氯化钙、氯化锰和氯化镁的影响更明显。电泳分析结果表明,在75℃条件下含有组蛋白变体H2A.Z的核小体的解聚速率明显低于常规核小体。采用荧光热漂移检测(fluorescence thermal shift analysis , FTS)进一步分析含有组蛋白变体H2A.Z核小体的稳定性,发现两类核小体的荧光信号均呈现2个明显的增长期,含有组蛋白变体H2A.Z核小体的第1个荧光信号增速期所对应的温度明显高于常规核小体,表明核小体中H2A.Z/H2B二聚体的解聚变性温度要高于常规的H2A/H2B二聚体,含有组蛋白变体H2A.Z核小体的热稳定性高。研究结果均表明,含有组蛋白变体H2A.Z的核小体的结构比常规核小体的结构稳定。     

Activation of gamma-aminobutyric acid insensitive chloride channels in mouse brain synaptic vesicles by avermectin B1a.

The interaction of avermectin B1a (AVMB1a) with mouse brain chloride channels was characterized using a radiochloride efflux assay. The loss of intravesicular chloride from synaptoneurosomes preloaded with 36Cl involved an initial rapid phase followed by a slower phase that approached equilibrium within 10 min. AVMB1a stimulated a 30% loss of intravesicular chloride within the first 2 s of exposure; however, AVMB1a had no effect on the rate of the slower phase of chloride loss. Experiments with lysed synaptoneurosomes showed that both chloride loading and basal and AVMB1a-stimulated chloride release required the presence of intact vesicles. The efflux of 36Cl from mouse brain synaptosomes and the stimulation of efflux by AVMB1a were qualitatively similar to the results obtained with synaptoneurosomes but involved much lower overall levels of chloride loading and release. AVMB1a produced half-maximal stimulation of chloride efflux from synaptoneurosomes at a concentration of 2.1 +/- 0.3 microM and a 35.4 +/- 1.4% maximal loss of intravesicular chloride at saturating concentrations. gamma-Aminobutyric acid (GABA), bicuculline, or the chloride channel blockers picrotoxinin, t-butylbicyclophosphorothionate (TBPS) 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS), and anthracene 9-carboxylic acid (9-CA) had little or no effect on the loss of chloride from synaptoneurosomes either in the presence or the absence of AVMB1a. However, the chlorinated cycloalkane insecticides dieldrin and lindane were equally effective as inhibitors of GABA-dependent chloride uptake and AVMB1a-stimulated chloride efflux. These data demonstrate that AVMB1a-stimulated chloride efflux from mouse brain synaptic vesicles results from the activation of GABA-insensitive chloride channels and that this action is distinct from their previously documented effects on GABA-gated chloride channels in mouse brain preparations. Our findings imply that both GABA-gated and GABA-insensitive chloride channels may be toxicologically significant targets for the action of avermectins.