The function of the chloride ion in photosynthetic oxygen evolution

The involvement of Cl(-) and several other monovalent anions in photosynthetic oxygen evolution was studied using photosystem II membranes depleted of Cl(-) by dialysis. The results of these studies differ significantly from results obtained using other depletion methods. Binding studies with glycerol as a cryoprotectant confirm our previous observations with sucrose of two interconvertible binding states of photosystem II with similar activities and with slow or fast exchange, respectively, of the bound ion. With glycerol, Cl(-) depletion decreased the oxygen evolution rate to 55% of that with Cl(-) present without decreasing the quantum efficiency of the reaction, supporting our previous conclusion that oxygen evolution can proceed at high rates in the absence of Cl(-). Further, after Cl(-) depletion the S(2) state multiline signal displayed the same periodic appearance with the same signal yield after consecutive laser flashes as with Cl(-) present. Br(-), I(-), and NO(3)(-), although with different capacities to reactivate oxygen evolution, also showed two binding modes. I(-) inhibited when bound in the low-affinity, fast-exchange mode but activated in the high-affinity mode. A comparison of the EPR properties of the S(2) state with these anions suggests that the nature of the ion or the binding mode only has a minor influence on the environment of the manganese. In contrast, F(-) completely inhibited oxygen evolution by preventing the S(2) to S(3) transition and shifted the equilibrium between the g = 4.1 and multiline S(2) forms toward the former, which suggests a considerable perturbation of the manganese cluster. To explain these and earlier observations, we propose that the role of chloride in the water-splitting mechanism is to participate together with charged amino acid side chains in a proton-relay network, which facilitates proton transfer from the manganese cluster to the medium. The structural requirements likely to be involved may explain the sensitivity of oxygen evolution to Cl(-) depletion or other perturbations.     

Clcn2 encodes the hyperpolarization-activated chloride channel in the ducts of mouse salivary glands

Transepithelial Cl(-) transport in salivary gland ducts is a major component of the ion reabsorption process, the final stage of saliva production. It was previously demonstrated that a Cl(-) current with the biophysical properties of ClC-2 channels dominates the Cl(-) conductance of unstimulated granular duct cells in the mouse submandibular gland. This inward-rectifying Cl(-) current is activated by hyperpolarization and elevated intracellular Cl(-) concentration. Here we show that ClC-2 immunolocalized to the basolateral region of acinar and duct cells in mouse salivary glands, whereas its expression was most robust in granular and striated duct cells. Consistent with this observation, nearly 10-fold larger ClC-2-like currents were observed in granular duct cells than the acinar cells obtained from submandibular glands. The loss of inward-rectifying Cl(-) current in cells from Clcn2(-/-) mice confirmed the molecular identity of the channel responsible for these currents as ClC-2. Nevertheless, both in vivo and ex vivo fluid secretion assays failed to identify significant changes in the ion composition, osmolality, or salivary flow rate of Clcn2(-/-) mice. Additionally, neither a compensatory increase in Cftr Cl(-) channel protein expression nor in Cftr-like Cl(-) currents were detected in Clcn2 null mice, nor did it appear that ClC-2 was important for blood-organ barrier function. We conclude that ClC-2 is the inward-rectifying Cl(-) channel in duct cells, but its expression is not apparently required for the ion reabsorption or the barrier function of salivary ductal epithelium.     

Identification of functional domains of the extrinsic 12 kDa protein in red algal PSII by limited rroteolysis and directed mutagenesis.

The extrinsic 12 kDa protein in red algal photosystem II (PSII) functions to minimize the chloride and calcium requirement of oxygen-evolving activity [Enami et al. (1998) Biochemistry 37: 2787]. In order to identify functional domains of the 12 kDa protein, we prepared the 12 kDa protein lacking N-terminal peptides or C-terminal peptides or both by limited proteolysis and directed mutagenesis. The resulting 12 kDa protein fragments were examined for their binding and functional properties by reconstitution experiments. (1) A peptide fragment from Gly-6 to C-terminus of the 12 kDa protein was prepared by V8 protease. This fragment rebound to PSII completely, and it reactivated oxygen evolution partially in the absence of Cl(-) and Ca(2+) ions but significantly in the presence of Cl(-) ion. (2) A peptide from Leu-10 to Phe-83 was obtained by chymotrypsin treatment. This peptide rebound to PSII effectively, but the rebinding did not restore oxygen evolution in both the absence and presence of Cl(-) and Ca(2+) ions. (3) Two mutant proteins, one lacking five residues and the other lacking nine residues of the N-terminus, were able to bind to PSII effectively. Recovery of oxygen evolution by their binding was almost the same as that reconstituted with the V8 protease-treated peptide. (4) Three mutant proteins lacking ten, seven or three residues of the C-terminus effectively rebound to PSII, but their binding did not result in recovery of the oxygen evolution. In contrast, reconstitution with a mutant protein lacking one residue of the C-terminus showed the same high restoration of oxygen evolution as reconstitution with the full-length 12 kDa protein. (5) These results indicate that two residues from lysine of the C-terminus of the 12 kDa protein constitute an important domain for minimizing the chloride and calcium requirement of oxygen evolution. In addition, the N-terminus of the protein, at least five residues, has a secondary function for the chloride requirement.     

Polymerase chain reaction-based mutageneses identify key transporters belonging to multigene families involved in Na+ and pH homeostasis of Synechocystis sp. PCC 6803

Primary ion pumps and antiporters exist as multigene families in the Synechocystis sp. PCC 6803 genome and show very strong homologies to those found in higher plants. The gene knock-outs of five putative Na+/H+ antiporters (slr1727, sll0273, sll0689, slr1595 and slr0415) and seven cation ATPases (sll1614, sll1920, slr0671-72, slr0822, slr1507-08-09, slr1728- 29 and slr1950) in the model cyanobacterium (http://www.kazusa.or.jp/cyano/cyano.html) were performed in this study relying on homologous recombination with mutagenenic fragments constructed using a fusion polymerase chain reaction (PCR) approach. The impacts of these gene knock-outs were evaluated in terms of Na+ and pH, and light-induced acidification and alkalization that are asso-ciated with inorganic carbon uptake. Two of the five putative antiporter mutants exhibit a characteristic interplay between the pH and Na+ dependence of growth, but only one of the antiporters appears to be necessary for high NaCl tolerance. On the other hand, the mutation of one of the two copper-trafficking ATPases produces a cell line that shows acute NaCl sensitivity. Additionally, disruptions of a putative Ca2+-ATPase and a gene cluster encoding a putative Na+-ATPase subunit also cause high NaCl sensitivity. The findings and possible mechanisms are discussed in relation to the potential roles of these transporters in Synechocystis sp. PCC 6803.     

A novel protein involved in the functional assembly of the oxygen-evolving complex of photosystem II in Synechocystis sp. PCC 6803

Mutation of Glu69 to Gln in the D2 protein of photosystem II is known to lead to a loss of photoautotrophic growth in Synechocystis sp. PCC 6803. However, second-site mutants (pseudorevertants) with restored photoautotrophic growth but still maintaining the E69Q mutation in D2 are easily obtained. Using a genomic mapping technique involving functional complementation, the secondary mutation was mapped to slr0286 in two independent mutants. The mutations in Slr0286 were R42M or R394H. To study the function of Slr0286, mutants of E69Q and of the wild-type strain were made that lacked slr0286. Deletion of slr0286 did not affect photoautotrophic capacity in wild type but led to a marked decrease in the apparent affinity of Ca(2+) to its binding site at the water-splitting system of photosystem II and to a reduced heat tolerance of the oxygen-evolving system, particularly in E69Q. Moreover, a small increase in the half-time for photoactivation of the oxygen-evolving complex of photosystem II for both wild type and the E69Q mutant was observed in the absence of Slr0286. The accumulation of photosystem II reaction centers, dark stability of the oxygen-evolving apparatus, stability of oxygen evolution, and the kinetics of charge recombination between Q(A)(-) and the donor side were not affected by deletion of slr0286. Slr0286 lacks clear functional motifs, and no homologues are apparent in other organisms, even not in other cyanobacteria. In any case, Slr0286 appears to help the functional assembly and stability of the water-splitting system of photosystem II.     

Structural insights into chloride and proton-mediated gating of CLC chloride channels

CLC Cl(-) channels fulfill numerous physiological functions as demonstrated by their involvement in several human genetic diseases. They have an unusual homodimeric architecture in which each subunit forms an individual pore whose open probability is regulated by various physicochemical factors, including voltage, Cl(-) concentration, and pH. The voltage dependence of Torpedo channel CLC-0 is derived probably indirectly from the translocation of a Cl(-) ion through the pore during the opening step. Recent structure determinations of bacterial CLC homologues marked a breakthrough for the structure-function analysis of CLC channels. The structures revealed a complex fold with 18 alpha-helices and two Cl(-) ions per subunit bound in the center of the protein. The side chain of a highly conserved glutamate residue that resides in the putative permeation pathway appears to be a major component of the channel gate. First studies have begun to exploit the bacterial structures as guides for a rational structure-function analysis. These studies confirm that the overall structure seems to be conserved from bacteria to humans. A full understanding of the mechanisms of gating of eukaryotic CLC channels is, however, still lacking.     

Chloride cofactor in the photosynthetic oxygen-evolving complex studied by fourier transform infrared spectroscopy

Fourier transform infrared (FTIR) spectroscopy, using midfrequency S2/S1 FTIR difference spectra, has been applied to studies of chloride cofactor in the photosynthetic oxygen-evolving complex (OEC) to determine the effects of Cl(-) depletion and monovalent anion substitution. Cl(-) depletion resulted in the disappearance of a large part of the amide I and II vibrational modes, and induced characteristic modification in the features of the stretching modes of the carboxylate ligands of the Mn cluster. The normal spectral features were largely restored by replenishment of Cl(-) except for some changes in amide bands. The overall features of Br(-) -, I(-) -, or NO3(-) -substituted spectra were similar to those of the Cl(-) -reconstituted spectrum, consistent with their ability to support oxygen evolution. In contrast, the spectrum was significantly altered by the replacement of Cl(-) with F- or CH3COO(-), which resulted in marked suppression and distortion of both the carboxylate and amide bands. The activity of oxygen evolution restored by NO3(-) was as high as that by Cl(-) when measured under limited light conditions, indicating that the NO3(-) -substituted OEC is fully active in oxygen evolution, although with a slow turnover rate. The double-difference spectrum between the 14NO3(-) -substituted and 15NO3- -substituted S2/S1 difference spectrum showed isotopic bands for asymmetric NO stretching mode in the region of 1400-1300 cm(-1) due to NO3(-) bound to the Cl(-) site. This demonstrated structural coupling between the Cl(-) site and the Mn cluster. A proposed model for the isotopic bands suggested that Cl(-) as well as NO3(-) is not directly associated with the Mn cluster and exists in a more symmetric configuration and weaker binding state in the S2 state than in the S1 state. These results also suggest that Cl(-) is required for changes in the structure of the specific carboxylate ligand of the Mn cluster as well as the peptide backbone of protein matrixes upon the transition from S1 to S2.     

Characterization of a sodium-regulated glutaminase from cyanobacterium Synechocystis sp. PCC 6803

Glutaminase is widely distributed among microorganisms and mammals with important functions. Little is known regarding the biochemical properties and functions of the deamidating enzyme glutaminase in cyanobacteria. In this study a putative glutaminase encoded by gene slr2079 in Synechocystis sp. PCC 6803 was investigated. The slr2079 was expressed as histidine-tagged fusion protein in Escherichia coli. The purified protein possessed glutaminase activity, validating the functional assignment of the genomic annotation. The apparent K _m value of the recombinant protein for glutamine was 26.6 ± 0.9 mmol/L, which was comparable to that for some of other microbial glutaminases. Analysis of the purified protein revealed a two-fold increase in catalytic activity in the presence of 1 mol/L Na⁺. Moreover, the K _m value was decreased to 12.2 ± 1.9 mmol/L in the presence of Na⁺. These data demonstrate that the recombinant protein Slr2079 is a glutaminase which is regulated by Na⁺ through increasing its affinity for substrate glutamine. The slr2079 gene was successfully disrupted in Synechocystis by targeted mutagenesis and the Δslr2079 mutant strain was analyzed. No differences in cell growth and oxygen evolution rate were observed between Δslr2079 and the wild type under standard growth conditions, demonstrating slr2079 is not essential in Synechocystis. Under high salt stress condition, however, Δslr2079 cells grew 1.25-fold faster than wild-type cells. Moreover, the photosynthetic oxygen evolution rate of Δslr2079 cells was higher than that of the wild-type. To further characterize this phenotype, a number of salt stress-related genes were analyzed by semi-quantitative RT-PCR. Expression of gdhB and prc was enhanced and expression of desD and guaA was repressed in Δslr2079 compared to the wild type. In addition, expression of two key enzymes of ammonium assimilation in cyanobacteria, glutamine synthetase (GS) and glutamate synthase (GOGAT) was examined by semi-quantitative RT-PCR. Expression of GOGAT was enhanced in Δslr2079 compared to the wild type while GS expression was unchanged. The results indicate that slr2079 functions in the salt stress response by regulating the expression of salt stress related genes and might not play a major role in glutamine breakdown in Synechocystis. Supported by the National Natural Sciences Foundation of China (Grant No. 30500108) and Hundred Talents Program of Chinese Academy of Sciences.     

Multiple potassium and chloride channels in the human colon carcinoma cell line SW1116

SW1116 cells have a profound capacity for secreting mucin molecules bearing the Lewisa epitope. Mucin molecules with the same epitope have been found to be elevated in the serum of patients with cystic fibrosis, a disease with defective ion channels. We therefore decided to study ion channels in this cell line. In the present work, we report the presence of two K(+)-channels and two Cl(-)-channels in the apical membrane of SW1116 cells. One of the K(+)-channels has a large conductance (approximately 278 pS), anomalous rectifying properties, and is inactivated rapidly. The second type exhibited a linear I/V curve (19 pS), was voltage insensitive and inactivation was not observed. In cell-attached patches, spontaneous openings of chloride channels were seen with higher frequency than previously reported in other colon carcinoma cell lines or airway epithelial cells. Inside-out experiments allowed identification of two different Cl(-)-channels (Cl(-)-1 and Cl(-)-2). Both exhibited rectification, but in opposite directions, and both were insensitive to NIPAB.     

Positive charges at the intracellular mouth of the pore regulate anion conduction in the CFTR chloride channel

Many different ion channel pores are thought to have charged amino acid residues clustered around their entrances. The so-called surface charges contributed by these residues can play important roles in attracting oppositely charged ions from the bulk solution on one side of the membrane, increasing effective local counterion concentration and favoring rapid ion movement through the channel. Here we use site-directed mutagenesis to identify arginine residues contributing important surface charges in the intracellular mouth of the cystic fibrosis transmembrane conductance regulator (CFTR) Cl(-) channel pore. While wild-type CFTR was associated with a linear current-voltage relationship with symmetrical solutions, strong outward rectification was observed after mutagenesis of two arginine residues (R303 and R352) located near the intracellular ends of the fifth and sixth transmembrane regions. Current rectification was dependent on the charge present at these positions, consistent with an electrostatic effect. Furthermore, mutagenesis-induced rectification was more pronounced at lower Cl(-) concentrations, suggesting that these mutants had a reduced ability to concentrate Cl(-) ions near the inner pore mouth. R303 and R352 mutants exhibited reduced single channel conductance, especially at negative membrane potentials, that was dependent on the charge of the amino acid residue present at these positions. However, the very low conductance of both R303E and R352E-CFTR could be greatly increased by elevating intracellular Cl(-) concentration. Modification of an introduced cysteine residue at position 303 by charged methanethiosulfonate reagents reproduced charge-dependent effects on current rectification. Mutagenesis of arginine residues in the second and tenth transmembrane regions also altered channel permeation properties, however these effects were not consistent with changes in channel surface charges. These results suggest that positively charged arginine residues act to concentrate Cl(-) ions at the inner mouth of the CFTR pore, and that this contributes to maximization of the rate of Cl(-) ion permeation through the pore.     

Characterization of a sodium-regulated glutaminase from cyanobacterium <Emphasis Type="Italic">Synechocystis</Emphasis> sp. PCC 6803

Glutaminase is widely distributed among microorganisms and mammals with important functions. Little is known regarding the biochemical properties and functions of the deamidating enzyme glutaminase in cyanobacteria. In this study a putative glutaminase encoded by gene slr2079 in Synechocystis sp. PCC 6803 was investigated. The slr2079 was expressed as histidine-tagged fusion protein in Escherichia coli. The purified protein possessed glutaminase activity, validating the functional assignment of the genomic annotation. The apparent K _m value of the recombinant protein for glutamine was 26.6 ± 0.9 mmol/L, which was comparable to that for some of other microbial glutaminases. Analysis of the purified protein revealed a two-fold increase in catalytic activity in the presence of 1 mol/L Na⁺. Moreover, the K _m value was decreased to 12.2 ± 1.9 mmol/L in the presence of Na⁺. These data demonstrate that the recombinant protein Slr2079 is a glutaminase which is regulated by Na⁺ through increasing its affinity for substrate glutamine. The slr2079 gene was successfully disrupted in Synechocystis by targeted mutagenesis and the Δslr2079 mutant strain was analyzed. No differences in cell growth and oxygen evolution rate were observed between Δslr2079 and the wild type under standard growth conditions, demonstrating slr2079 is not essential in Synechocystis. Under high salt stress condition, however, Δslr2079 cells grew 1.25-fold faster than wild-type cells. Moreover, the photosynthetic oxygen evolution rate of Δslr2079 cells was higher than that of the wild-type. To further characterize this phenotype, a number of salt stress-related genes were analyzed by semi-quantitative RT-PCR. Expression of gdhB and prc was enhanced and expression of desD and guaA was repressed in Δslr2079 compared to the wild type. In addition, expression of two key enzymes of ammonium assimilation in cyanobacteria, glutamine synthetase (GS) and glutamate synthase (GOGAT) was examined by semi-quantitative RT-PCR. Expression of GOGAT was enhanced in Δslr2079 compared to the wild type while GS expression was unchanged. The results indicate that slr2079 functions in the salt stress response by regulating the expression of salt stress related genes and might not play a major role in glutamine breakdown in Synechocystis.     

Intracellular chloride activity in cultured mesangial cells.

Glomerular mesangial cells require Cl ions for the development of a variety of metabolic and functional properties. In the present study the electrochemical distribution for Cl- was examined in cultured rat mesangial cells with Cl(-)-sensitive intracellular microelectrodes. It was determined that the intracellular Cl activity exceeded the levels predicted for a passively distributed ion. This was further substantiated by exposing mesangial cells to 10(-5) M bumetanide which drove intracellular Cl to a value close to electrochemical equilibrium. We conclude that Cl accumulates in mesangial cells, against its electrochemical gradient, through a transport pathway that is highly sensitive to bumetanide.     

Hypoosmotic stimuli activate a chloride conductance in rat taste cells

The oral cavity is subjected to a wide range of osmotic conditions, yet little is known about how solution osmolarity affects performance of the gustatory system. In order to elucidate the mechanism by which hypoosmotic stimuli affect the peripheral taste system, I have attempted to characterize the effects of hypoosmotic stimuli on individual rat taste receptor cells (TRCs) using whole-cell patch clamp recording. Currents elicited in response to voltage ramps (-90 to +60 mV) were recorded in control saline and in solutions varying only in osmolarity (-30, -60 and -90 mOsm). In roughly two-thirds of cells, hypoosmotic solutions (230 mOsm) caused a 15% increase in cell capacitance and activated a reversible conductance that exhibited marked adaptation in the continued presence of the stimulus. Similar responses could be elicited in taste cells from taste buds in the foliate and vallate papillae, the soft palate, the nasopharynx and the epiglottis. Ion substitution experiments were consistent with the interpretation that the predominant ion carried through these apparent volume- or stretch-activated channels was Cl(-) under normal conditions. Reversal potentials for the hypoosmotic-induced current closely matched those predicted by the Goldman-Hodgkin-Katz constant field equation for a Cl(-) conductance. The relative permeability sequence of the hypoosmotic-activated current in TRCs was thiocyanate(-) > or = l(-) > or = Br(-) > Cl(-) > or = F(-) > or = isethionate(-) > gluconate(-). Pharmacological experiments revealed that this Cl(-) conductance was inhibited by 4,4'-diisothiocyanatostilbene-2, 2'-disulfonic acid and 5-nitro-3-(3-phenyl-propylamino)benzoic acid (EC(50) = 1.3 and 4.6 microM, respectively), but not by CdCl(2) (300 microM) nor GdCl(3) (200 microM). I hypothesize that this hypoosmotic-activated Cl(-) conductance, which is similar to the well-characterized swelling-activated Cl(-) current, may contribute to volume regulation and could represent the transduction mechanism by which the presence of hypoosmotic stimuli, including water, may be signaled in taste receptor cells.     

Activation of potassium and chloride channels by tumor necrosis factor alpha. Role in liver cell death

Despite abundant evidence for changes in mitochondrial membrane permeability in tumor necrosis factor (TNF)-mediated cell death, the role of plasma membrane ion channels in this process remains unclear. These studies examine the influence of TNF on ion channel opening and death in a model rat liver cell line (HTC). TNF (25 ng/ml) elicited a 2- and 5-fold increase in K(+) and Cl(-) currents, respectively, in HTC cells. These increases occurred within 5-10 min after TNF exposure and were inhibited either by K(+) or Cl(-) substitution or by K(+) channel blockers (Ba(2+), quinine, 0.1 mm each) or Cl(-) channel blockers (10 microm 5-nitro-2-(3-phenylpropylamino)benzoic acid and 0.1 mm N-phenylanthranilic acid), respectively. TNF-mediated increases in K(+) and Cl(-) currents were each inhibited by intracellular Ca(2+) chelation (5 mm EGTA), ATP depletion (4 units/ml apyrase), and the protein kinase C (PKC) inhibitors chelerythrine (10 micrometer) or PKC 19-36 peptide (1 micrometer). In contrast, currents were not attenuated by the calmodulin kinase II 281-309 peptide (10 micrometer), an inhibitor of calmodulin kinase II. In the presence of actinomycin D (1 micrometer), each of the above ion channel blockers significantly delayed the progression to TNF-mediated cell death. Collectively, these data suggest that activation of K(+) and Cl(-) channels is an early response to TNF signaling and that channel opening is Ca(2+)- and PKC-dependent. Our findings further suggest that K(+) and Cl(-) channels participate in pathways leading to TNF-mediated cell death and thus represent potential therapeutic targets to attenuate liver injury from TNF.     

Mitochondria-rich cells in anuran amphibia: chloride conductance and regional distribution over the body surface

The distribution and density (D(mrc)) of mitochondria-rich cells (MR cells) in skin epithelium, were determined over the whole body surface in nine species of anuran Amphibia that live in a variety of habitats. It was found that the more terrestrial species (beginning with Hyla arborea) have a higher density of MR cells in their pelvic region. In the skin of aquatic (Xenopus laevis) or fossorial (Pelobates syriacus) species, D(mrc) is evenly distributed over the whole body surface. In dorsal skin pieces of H. arborea that lack detectable MR cells, transepithelial voltage activation did not induce Cl(-) conductance as it did in ventral pieces. Skins from Bufo viridis and X. laevis, both have MR cells in their skin, differ markedly in their biophysical properties: a Cl(-) specific current conductance is predominant in the skin epithelium of B. viridis, and is absent in X. laevis. In the latter, anionic conductance is due to glandular secretion. The biophysical properties cannot therefore be related solely to the presence or density of MR cells. Mitochondria-rich cells are sites of Cl(-) conductance across the skin of those amphibians that show this property, but must have different function(s) in other species. It is suggested that the specific zonal distribution of MR cells in the species that were examined in this study could be due to ion exchange activity and water conservation in more terrestrial environments.     

Hydrogen-bonding alterations of the protonated Schiff base and water molecule in the chloride pump of Natronobacterium pharaonis

Halorhodopsin is a light-driven chloride ion pump. Chloride ion is bound in the Schiff base region of the retinal chromophore, and unidirectional chloride transport is probably enforced by the specific hydrogen-bonding interaction with the protonated Schiff base and internal water molecules. In this article, we study hydrogen-bonding alterations of the Schiff base and water molecules in halorhodopsin of Natronobacterium pharaonis (pHR) by assigning their N-D and O-D stretching vibrations in D(2)O, respectively. Highly accurate low-temperature Fourier transform infrared spectroscopy revealed that hydrogen bonds of the Schiff base and water molecules are weak in the unphotolyzed state, whereas they are strengthened upon retinal photoisomerization. Halide dependence of the stretching vibrations enabled us to conclude that the Schiff base forms a direct hydrogen bond with Cl(-) only in the K intermediate. Hydrogen bond of the Schiff base is further strengthened in the L(1) intermediate, whereas the halide dependence revealed that the acceptor is not Cl(-), but presumably a water molecule. Thus, it is concluded that the hydrogen-bonding interaction between the Schiff base and Cl(-) is not a driving force of the motion of Cl(-). Rather, the removal of its hydrogen bonds with the Schiff base and water(s) makes the environment around Cl(-) less polar in the L(1) intermediate, which presumably drives the motion of Cl(-) from its binding site to the cytoplasmic domain.     

Fluorescence measurement of chloride transport in monolayer cultured cells. Mechanisms of chloride transport in fibroblasts.

The methodology has been developed to measure Cl activity and transport in cultured cells grown on a monolayer using the entrapped Cl-sensitive fluorophore 6-methoxy-N-[3-sulfopropyl] quinolinium (SPQ). The method was applied to a renal epithelial cell line, LLC-PKI, and a nonepithelial cell line, Swiss 3T3 fibroblasts. SPQ was nontoxic to cells when present for greater than h in the culture media. To load with SPQ (5 mM), cells were made transiently permeable by exposure to hypotonic buffer (150 mOsm, 4 min). Intracellular fluorescence was monitored continuously by epifluorescence microscopy using low illumination intensity at 360 +/- 5 nm excitation wavelength and photomultiplier detection at greater than 410 nm. Over 60 min at 37 degrees C, there was no photobleaching and less than 10% leakage of SPQ out of cells; intracellular SPQ fluorescence was uniform. SPQ fluorescence was calibrated against intracellular [Cl] using high K solutions containing the ionophores nigericin and tributyltin. The Stern-Volmer constant (Kq) for quenching of intracellular SPQ by Cl was 13 M-1 for fibroblasts and LLC-PKl cells. In the absence of Cl, SPQ lifetime was 26 ns in aqueous solution and 3.7 +/- 0.6 ns in cells, showing that the lower Kq in cells than in free solution (Kq = 118 M-1) was due to SPQ quenching by intracellular anions. To examine Cl transport mechanisms, the time course of intracellular [Cl] was measured in response to rapid Cl addition and removal in the presence of ion or pH gradients. In fibroblasts, three distinct Cl transporting systems were identified: a stilbeneinhibitable Cl/HCO3 exchanger, a furosemide-sensitive Na/K/2Cl cotransporter, and a Ca-regulated Cl conductance. These results establish a direct optical method to measure intracellular [Cl] continuously in cultured cells.     

光系统Ⅱ颗粒的毫秒延迟荧光的研究

本文主要报导了具有放氧活性的光系统Ⅱ(PSⅡ)颗粒的毫秒延迟荧光(ms-DF)的特性以及NH_4Cl对它的调节作用.