Identification by site-directed mutagenesis of three arginines in uncoupling protein that are essential for nucleotide binding and inhibition.
AuthorsModrianský M, Murdza-Inglis DL, Patel HV, Freeman KB, Garlid KD
JournalJ Biol Chem
PubMed ID9312070
Primary regulation of uncoupling protein is mediated by purine nucleotides, which bind to the protein and allosterically inhibit fatty acid-induced proton transport. To gain increased understanding of nucleotide regulation, we evaluated the role of basic amino acid residues using site-directed mutagenesis. Mutant and wild-type proteins were expressed in yeast, purified, ... More
Fluorescent indicators of ion concentrations.
AuthorsTsien RY
JournalMethods Cell Biol
PubMed ID2538708
Efficient endocytosis of the cystic fibrosis transmembrane conductance regulator requires a tyrosine-based signal.
'We previously demonstrated that the cystic fibrosis transmembrane conductance regulator (CFTR) is rapidly endocytosed in epithelial cells (Prince, L. S., Workman, R. B., Jr., and Marchase, R. B. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 5192-5196). To determine the structural features of CFTR required for endocytosis, we prepared ... More
Estimation of intracellular chloride activity in isolated perfused rabbit proximal convoluted tubules using a fluorescent indicator.
AuthorsKrapf R, Berry CA, Verkman AS
JournalBiophys J
PubMed ID3395662
'The methodology has been developed to measure cell chloride activity by fluorescence microscopy using the chloride-sensitive dye, 6-methoxy-1-(3-sulfonatopropyl)quinolinium (SPQ). SPQ was loaded into cells of the in vitro microperfused rabbit proximal convoluted tubule by a 10 min luminal perfusion with 20 mM SPQ at 38 degrees C. Fluorescence was excited ... More
Regulation of Cl/HCO3 exchange in gastric parietal cells.
AuthorsThomas HA, Machen TE
JournalCell Regul
PubMed ID1742343
'Microspectrofluorimetry of the fluorescent indicators 2',7'-bis-(2-carboxyethyl)-5(and-6)carboxyfluorescein and 6-methoxy-N-(3-sulfopropyl)-quinolinium was used to measure intracellular pH (pHi), intracellular Cl (Cli), and transmembrane fluxes of HCO3 and Cl in single parietal cells (PC) in isolated rabbit gastric glands incubated in HCO3/CO2-buffered solutions. Steady-state pHi was 7.2 in both resting (50 microM cimetidine) and ... More
The cytosolic chloride concentration in macula densa and cortical thick ascending limb cells.
AuthorsSalomonsson M, Gonzalez E, Kornfeld M, Persson AE
JournalActa Physiol Scand
PubMed ID8386427
'It is believed that chloride transport through the macula densa (MD) cells is a factor involved in the tubuloglomerular feedback (TGF) mechanism and in MD-mediated renin release. In this study isolated and perfused rabbit kidney cortical thick ascending limb (cTAL) segments containing MD plaques and attached glomeruli were loaded with ... More
Mechanism of uptake of the fluorescent dye 2-(4-dimethylaminostyryl)-1-ethylpyridinium cation (DMP+) by phospholipid vesicles.
AuthorsSedgwick EG, Bragg PD
JournalBiochim Biophys Acta
PubMed ID8443217
'The fluorescent dye 2-(4-dimethylaminostyryl)-1-ethylpyridinium cation (DMP+) is taken up by liposomes of egg phosphatidylcholine in response to the imposition of a transmembrane potential. Entry of DMP+ into the bilayer driven by the transmembrane potential is accompanied by a change in the fluorescence emission maximum of the dye. This change reflects ... More
On the mechanism of GABA-induced currents in cultured rat cortical neurons.
AuthorsDallwig R, Deitmer JW, Backus KH
JournalPflugers Arch
PubMed ID9929572
'We applied the perforated-patch-clamp technique to cultured cortical neurons of the rat to characterize the ionic basis of membrane potential changes and membrane currents induced by gamma-aminobutyric acid (GABA). Gramicidin was used as the membrane-perforating agent, to allow the recording of whole-cell currents without impairing the intracellular Cl- concentration ([Cl-]i). ... More
Swelling activated chloride channels in cultured bovine corneal endothelial cells.
AuthorsSrinivas SP, Guan Y, Bonanno JA
JournalExp Eye Res
PubMed ID10068482
'Swelling induced enhancement of anion permeability was investigated using the halide-sensitive fluorescent dye SPQ in cultured bovine corneal endothelial cells (BCEC). Rates of anion influx were quantified in terms of the rate of change of SPQ fluorescence during exposure to short duration pulses of Cl-, I-or NO3-while the cells were ... More
Assessment of swelling-activated Cl- channels using the halide-sensitive fluorescent indicator 6-methoxy-N-(3-sulfopropyl)quinolinium.
AuthorsSrinivas SP, Bonanno JA, Hughes BA
JournalBiophys J
PubMed ID9649372
'This study describes a quantitative analysis of the enhancement in anion permeability through swelling-activated Cl- channels, using the halide-sensitive fluorescent dye 6-methoxy-N-(3-sulfopropyl)quinolinium (SPQ). Cultured bovine corneal endothelial monolayers perfused with NO3- Ringer''s were exposed to I- pulses under isosmotic and, subsequently, hyposmotic conditions. Changes in SPQ fluorescence due to I- ... More
Halide fluxes in epithelial cells measured with an automated cell plate reader.
AuthorsMahlangu DA, Dix JA
JournalAnal Biochem
PubMed ID14715281
'A method is introduced to measure chloride permeability in cultured epithelial cells using 6-methoxy-N-(3-sulfopropyl)quinolinium (SPQ) and 6-methoxy-N-ethylquinolinium iodide quinolinium (MEQ) as fluorescent chloride-sensitive probes. The method involves growing cells in multiwell plates, incubating cells with SPQ or MEQ, and then exchanging intracellular or extracellular halide ions with nitrate. The resulting ... More
Polyethylenimine but not cationic lipids promotes transgene delivery to the nucleus in mammalian cells.
'The beta-galactosidase reporter gene, either free or complexed with various cationic vectors, was microinjected into mammalian cells. Cationic lipids but not polyethylenimine or polylysine prevent transgene expression when complexes are injected in the nucleus. Polyethylenimine and to a lesser extent polylysine, but not cationic lipids, enhance transgene expression when complexes ... More
Natural and azido fatty acids inhibit phosphate transport and activate fatty acid anion uniport mediated by the mitochondrial phosphate carrier.
AuthorsEngstová H, Zácková M, Ruzicka M, Meinhardt A, Hanus J, Krämer R, Jezek P
JournalJ Biol Chem
PubMed ID11085992
'The electroneutral P(i) uptake via the phosphate carrier (PIC) in rat liver and heart mitochondria is inhibited by fatty acids (FAs), by 12-(4-azido-2-nitrophenylamino)dodecanoic acid (AzDA) and heptylbenzoic acid ( approximately 1 microm doses) and by lauric, palmitic, or 12-azidododecanoic acids ( approximately 0.1 mm doses). In turn, reconstituted E. coli-expressed ... More
Mechanism of calcium transport stimulated by chlorothiazide in mouse distal convoluted tubule cells.
AuthorsGesek FA, Friedman PA
JournalJ Clin Invest
PubMed ID1322939
'Thiazide diuretics inhibit Na+ and stimulate Ca2+ absorption in renal distal convoluted tubules. Experiments were performed on immortalized mouse distal convoluted tubule (MDCT) cells to determine the mechanism underlying the dissociation of sodium from calcium transport and the stimulation of calcium absorption induced by thiazide diuretics. Control rates of 22Na+ ... More
Regulation of cAMP-dependent chloride channels in DC1 immortalized rabbit distal tubule cells in culture.
AuthorsRubera I, Tauc M, Verheecke-Mauze C, Bidet M, Poujeol C, Touret N, Cuiller B, Poujeol P
JournalAm J Physiol
PubMed ID9887086
'Cl- conductances were studied in an immortalized cell line (DC1) derived from rabbit distal bright convoluted tubule (DCTb). The DC1 clone was obtained after transfection of primary cultures of DCTb with pSV3 neo. RT-PCR experiments showed the presence of cystic fibrosis transmembrane conductance regulator (CFTR) mRNA in the DC1 cell ... More
Development and biological applications of chloride-sensitive fluorescent indicators.
AuthorsVerkman AS
JournalAm J Physiol
PubMed ID2205105
'Chloride movement across cell plasma and internal membranes, is of central importance for regulation of cell volume and pH, vectorial salt movement in epithelia, and, probably, intracellular traffic. Quinolinium-based chloride-sensitive fluorescent indicators provide a new approach to study chloride transport mechanisms and regulation that is complementary to 36Cl tracer methods, ... More
Measuring the adsorption of Fatty acids to phospholipid vesicles by multiple fluorescence probes.
AuthorsSimard JR, Kamp F, Hamilton JA,
JournalBiophys J
PubMed ID18296488
'Fatty acids (FA) are important nutrients that the body uses to regulate the storage and use of energy resources. The predominant mechanism by which long-chain fatty acids enter cells is still debated widely as it is unclear whether long-chain fatty acids require protein transporters to catalyze their transmembrane movement. We ... More
Intracellular chloride and calcium transients evoked by gamma-aminobutyric acid and glycine in neurons of the rat inferior colliculus.
AuthorsFrech MJ, Deitmer JW, Backus KH
JournalJ Neurobiol
PubMed ID10440738
'Microfluorometric recordings showed that the inhibitory neurotransmitters gamma-aminobutyric acid (GABA) and glycine activated transient increases in the intracellular Cl- concentration in neurons of the inferior colliculus (IC) from acutely isolated slices of the rat auditory midbrain. Current recordings in gramicidin-perforated patch mode disclosed that GABA and glycine mainly evoked inward ... More
Chloride-sensitive fluorescent indicators.
AuthorsGeddes CD, Apperson K, Karolin J, Birch DJ
JournalAnal Biochem
PubMed ID11373079
'Three fluorescent halide-sensitive quinolinium dyes have been produced by the reaction of the 6-methylquinoline heterocyclic nitrogen base with methyl bromide, methyl iodide, and 3-bromo-1-propanol. The quaternary salts, unlike the precursor molecule, are readily water soluble and the fluorescence intensity of these salts is reduced in the presence of aqueous chloride, ... More
Photoactivated azido fatty acid irreversibly inhibits anion and proton transport through the mitochondrial uncoupling protein.
AuthorsJezek P, Hanus J, Semrad C, Garlid KD
JournalJ Biol Chem
PubMed ID8626410
'The protonophoretic function of uncoupling protein (UCP) is activated by fatty acids. According to the "docking site" hypothesis (Jezek, P., and Garlid, K. D., J. Biol. Chem. 265, 19303-19311, 1990), the fatty acid binding site is identical with the anion channel of UCP. Skulachev (Skulachev, V. P. (1991) FEBS Lett. ... More
Hyperexpression of recombinant CFTR in heterologous cells alters its physiological properties.
AuthorsMohammad-Panah R, Demolombe S, Riochet D, Leblais V, Loussouarn G, Pollard H, Baró I, Escande D
JournalAm J Physiol
PubMed ID9486119
'We investigated whether high levels of expression of the cystic fibrosis transmembrane conductance regulator (CFTR) would alter the functional properties of newly synthesized recombinant proteins. COS-7, CFPAC-1, and A549 cells were intranuclearly injected with a Simian virus 40-driven pECE-CFTR plasmid and assayed for halide permeability using the 6-methoxy-N-(3-sulfopropyl)quinolinium fluorescent probe. ... More
Stimulation of Cl permeability in colonic crypts of Lieberkühn measured with a fluorescent indicator.
AuthorsHalm DR, Kirk KL, Sathiakumar KC
JournalAm J Physiol
PubMed ID8214063
'Crypts of Lieberkühn were isolated from rabbit distal colon and the halide-sensitive dye 6-methoxy-N-[3-sulfopropyl]quinolinium was used to monitor changes in cell Cl by fluorescence microscopy. Distal colon from rabbits actively secretes Cl and K when stimulated with prostaglandin (PG) E2 but secretes only K in response to epinephrine. The secretagogues ... More
Characterization of Cl- transport in vacuolar membrane vesicles using a Cl(-)-sensitive fluorescent probe: reaction kinetic models for voltage- and concentration-dependence of Cl- flux.
AuthorsPope AJ, Jennings IR, Sanders D, Leigh RA
JournalJ Membr Biol
PubMed ID2380980
'The effects of Cl- concentration and membrane potential (delta psi) on Cl- influx in isolated vesicles of vacuolar membrane (tonoplast) from red beet (Beta vulgaris L.) storage tissue have been characterized using the Cl(-)-sensitive fluorescent probe, 6-methoxy-1-(3-sulfonatopropyl)quinolinium (SPQ). The initial rate of Cl- transport into the vesicles was enhanced both ... More
The Na+2Cl-K+ cotransporter in the rectal gland of Squalus acanthias is activated by cell shrinkage.
AuthorsGreger R, Heitzmann D, Hug MJ, Hoffmann EK, Bleich M
JournalPflugers Arch
PubMed ID10370103
'Effects of cAMP on Cl- secretion, intracellular Cl- activity and cell volume were studied in isolated perfused rectal gland tubules (RGT) of Squalus acanthias with electrophysiological and fluorescence methods. Recording of equivalent short-circuit current (Isc) showed that cAMP stimulates Na+Cl- secretion in a biphasic manner. The first and rapid phase ... More
Use of colorimetric test strips for monitoring the effect of hemodialysis on salivary nitrite and uric acid in patients with end-stage renal disease: a proof of principle.
AuthorsBlicharz TM, Rissin DM, Bowden M, Hayman RB, DiCesare C, Bhatia JS, Grand-Pierre N, Siqueira WL, Helmerhorst EJ, Loscalzo J, Oppenheim FG, Walt DR,
JournalClin Chem
PubMed ID18676588
'BACKGROUND: Initial screening of potential biomarkers for monitoring dialysis was performed with saliva samples collected from patients with end-stage renal disease (ESRD). A more thorough analysis of the most promising markers identified in the initial screening was conducted with saliva samples acquired at hourly intervals throughout dialysis to monitor analyte ... More
Adenosine and its nucleotides activate wild-type and R117H CFTR through an A2B receptor-coupled pathway.
AuthorsClancy JP, Ruiz FE, Sorscher EJ
JournalAm J Physiol
PubMed ID9950763
'ATP and its metabolites stimulate Cl- secretion in human epithelium in vitro and in vivo. The specific purinergic receptor subtypes that govern these effects have been difficult to separate, in part due to multiple parallel pathways for Cl- secretion in respiratory and intestinal epithelia. In a simplified model using COS-7 ... More
Synthesis and characterization of dual-wavelength Cl--sensitive fluorescent indicators for ratio imaging.
AuthorsJayaraman S, Biwersi J, Verkman AS
JournalAm J Physiol
PubMed ID10070003
'The fluorescence of quinolinium-based Cl- indicators such as 6-methoxy-N-(3-sulfopropyl)quinolinium (SPQ) is quenched by Cl- by a collisional mechanism without change in spectral shape. A series of "chimeric" dual-wavelength Cl- indicators were synthesized by conjugating Cl--sensitive and -insensitive chromophores with spacers. The SPQ chromophore (N-substituted 6-methoxyquinolinium; MQ) was selected as the ... More
A flow cytometric method for measurement of intracellular chloride concentration in lymphocytes using the halide-specific probe 6-methoxy-N-(3-sulfopropyl) quinolinium (SPQ).
AuthorsPilas B, Durack G
JournalCytometry
PubMed ID9266752
'A flow cytometry method using the halide-specific fluorescent dye, 6-methoxy-N-(3-sulfopropyl) quinolinium (SPQ), has been developed to measure intracellular chloride concentration in single cells. Collisions with chloride quench the fluorescence of SPQ, making it possible to relate the measured fluorescence intensity to chloride concentration with a Stern-Volmer equation. To demonstrate the ... More
Ca2+/Na+ exchanger and Na+,K+ 2Cl- cotransporter in lens fiber plasma membrane vesicles.
AuthorsYe JJ, Zadunaisky JA
JournalExp Eye Res
PubMed ID1486938
'When the Ca(2+)-sensitive fluorescent probe, Fura-2, or the Na(+)-sensitive probe, SBFI, in their cell permeable forms or the Cl(-)-specific probe, SPQ, were incubated with plasma membrane vesicles prepared from dogfish and bovine lenses fibers, there was a selective accumulation of the ion-specific probes within the vesicles. The SBFI and Fura-2 ... More
Chloride secretion in kidney distal epithelial cells (A6) evoked by cadmium.
AuthorsFaurskov B, Bjerregaard HF
JournalToxicol Appl Pharmacol
PubMed ID10702366
'The effect of Cd(2+) on chloride secretion was examined in A6 renal epithelia cells by chloride-sensitive fluorescence (SPQ probe) and by the short-circuit-current (I(sc)) technique. Depleting the cells of Cl(-) suggests that the Cd(2+)-activated I(sc) (DeltaI(sc(Cd))) is dependent on the presence of Cl(-) ions. Among the Cl(-)-channel inhibitors the fenemates, ... More
Determination of GABA receptor-linked Cl- fluxes in rat cerebellar granule cells using a fluorescent probe SPQ.
AuthorsEngblom AC, Holopainen I, Akerman KE
JournalNeurosci Lett
PubMed ID2554225
'Gamma-Aminobutyric acid (GABA)-induced Cl- fluxes in cultured rat cerebellar granule cells were measured using the chloride-sensitive fluorescent probe SPQ (6-methoxy-N-(3-sulphopropyl)quinolinium) incorporated into the cells. The fluorescence of SPQ is quenched by Cl-ions. GABA and pentobarbitone increased the fluorescence of the probe when the Cl- gradient was directed outward by bathing ... More
A fluorimetric assay for the effects of cytolytic toxins on the transport properties of resealed erythrocyte ghosts.
AuthorsDalla Serra M, Pederzolli C, Menestrina G
JournalJ Biochem Biophys Methods
PubMed ID1491104
'We prepared resealed erythrocyte ghosts loaded with SPQ and chloride. We demonstrated that these membranes were still functional, as they were capable of exchanging anions, most probably through the band-3 protein. When cytolytic toxins (Escherichia coli hemolysin and Staphylococcus aureus alpha-toxin) were offered to the resealed ghosts, the internal SPQ ... More
Mechanism through which GABAA receptor modulates catecholamine secretion from bovine chromaffin cells.
AuthorsGonzalez MP, Oset-Gasque MJ, Castro E, Bugeda J, Arce C, Parramon M
JournalNeuroscience
PubMed ID1322511
'The actions and mechanism of GABAergic modulation of catecholamine secretion from isolated bovine chromaffin cells were investigated. The GABAA receptor agonist muscimol induced a fast rise in cytosolic [Ca2+]. The mean peak increase was 290 +/- 30 nM over basal levels. The increase in cytosolic [Ca2+] was abolished in the ... More
Direct measurement of nitrite transport across erythrocyte membrane vesicles using the fluorescent probe, 6-methoxy-N-(3-sulfopropyl) quinolinium.
AuthorsShingles R, Roh MH, McCarty RE
JournalJ Bioenerg Biomembr
PubMed ID9559862
'Nitrite was shown to quench the fluorescence of 6-methoxy-N-(3-sulfopropyl) quinolinium (SPQ) almost twofold more than chloride. SPQ loaded inside vesicles prepared from asolectin and isolated erythrocyte ghosts allowed for the direct measurement of nitrite movement across these membranes. Movement of nitrite across asolectin occurred by diffusion as HNO2 in a ... More
Effects of KCNQ1 channel blocker, 293B, on the acetylcholine-induced Cl- secretion of rat pancreatic acini.
AuthorsLee JE, Park HS, Uhm DY, Kim SJ
JournalPancreas
PubMed ID15097862
'In rat pancreatic acini (RPAs), acetylcholine (ACh) typically induces a tonic depolarization of membrane potential (Vm) via increasing cytoplasmic Ca2+ concentration and subsequent activation of Cl- channels. In this study, to investigate the role of K+ channels during the ACh-induced Cl- secretion, the intracellular Cl- concentration ([Cl-]i) of RPAs was ... More
beta3-adrenoceptor control the cystic fibrosis transmembrane conductance regulator through a cAMP/protein kinase A-independent pathway.
AuthorsLeblais V, Demolombe S, Vallette G, Langin D, Baró I, Escande D, Gauthier C
JournalJ Biol Chem
PubMed ID10037693
'In human cardiac myocytes, we have previously identified a functional beta3-adrenoceptor in which stimulation reduces action potential duration. Surprisingly, in cardiac biopsies obtained from cystic fibrosis patients, beta3-adrenoceptor agonists produced no effects on action potential duration. This result suggests the involvement of cystic fibrosis transmembrane conductance regulator (CFTR) chloride current ... More
Oligomerization of the channel-forming toxin aerolysin precedes insertion into lipid bilayers.
Authorsvan der Goot FG, Pattus F, Wong KR, Buckley JT
JournalBiochemistry
PubMed ID7680572
'Oligomerization is a necessary step in channel formation by the bacterial toxin aerolysin. We have identified a region of aerolysin containing two tryptophans which influence the ability of the protein to oligomerize. Changing the tryptophan at position 371 or 373 to leucine resulted in mutant proteins that oligomerized at much ... More
Alkylsulfonates as probes of uncoupling protein transport mechanism. Ion pair transport demonstrates that direct H(+) translocation by UCP1 is not necessary for uncoupling.
AuthorsJaburek M, Varecha M, Jezek P, Garlid KD
JournalJ Biol Chem
PubMed ID11468281
'The mechanism of fatty acid-dependent uncoupling by mitochondrial uncoupling proteins (UCP) is still in debate. We have hypothesized that the anionic fatty acid head group is translocated by UCP, and the proton is transported electroneutrally in the bilayer by flip-flop of the protonated fatty acid. Alkylsulfonates are useful as probes ... More
Kinetics of chloride-bicarbonate exchange across the human red blood cell membrane.
AuthorsGreco FA, Solomon AK
JournalJ Membr Biol
PubMed ID9312209
'We use a fluorescent probe of [Cl-], 6-methoxy-N-(3-sulfopropyl)quinolinium (SPQ), to study Cl-/HCO-3 exchange in human erythrocyte ghosts in a stopped-flow apparatus at 4 degrees C. The quench constant of SPQ in our Cl-/HCO-3/HPO=4 system at pH 7.4 is 0.065 +/- 0.005 mM-1. The time course of Cl-/HCO-3 exchange does not ... More
Intracellular ramification of endothelin signal.
AuthorsIijima K, Lin L, Nasjletti A, Goligorsky MS
JournalAm J Physiol
PubMed ID2035621
'Effects of porcine 1-21 endothelin (ET-1) on [Ca2+]i, [Na+]i, and [Cl-]i and on membrane potential were studied in individual mesangial (MC) and vascular smooth muscle (VSMC) cells using microspectrofluorimetry of fura-2, SBFI, SPQ, and bis-oxonol, respectively. ET-1 increased [Ca2+]i by fivefold, showing an immediate and a sustained phase of response. ... More
Endogenous nitric oxide inhibits chloride transport in the thick ascending limb.
AuthorsPlato CF, Stoos BA, Wang D, Garvin JL
JournalAm J Physiol
PubMed ID9887091
'Nitric oxide (NO) inhibits transport in various nephron segments, and the thick ascending limb of the loop of Henle (TALH) expresses NO synthase (NOS). However, the effects of NO on TALH transport have not been extensively studied. We hypothesized that endogenously produced NO directly decreases NaCl transport by the TALH. ... More
Generation of cAMP-activated chloride currents by expression of CFTR.
AuthorsAnderson MP, Rich DP, Gregory RJ, Smith AE, Welsh MJ
JournalScience
PubMed ID1704151
'Mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) cause cystic fibrosis. In order to evaluate its function, CFTR was expressed in HeLa, Chinese hamster ovary (CHO), and NIH 3T3 fibroblast cells, and anion permeability was assessed with a fluorescence microscopic assay and the whole-cell patch-clamp technique. Adenosine 3'',5''-monophosphate (cAMP) ... More
Regulation of intracellular chloride concentration in rat lactotrophs: possible role of mitochondria.
AuthorsGarcia L, Rigoulet M, Georgescauld D, Dufy B, Sartor P
JournalFEBS Lett
PubMed ID9000524
'Increasing evidence is accumulating for the involvement of chloride ions in the stimulus-secretion coupling of pituitary cells. We show that the mean intracellular chloride concentration [Cl-]i of rat lactotroph cells maintained in culture is high, close to 60 mM (59.4 mM), using the Cl- sensitive fluorescent probe 6-methoxy-N-(3-sulfopropyl) quinolinium (SPQ), ... More
The effect of mechanical deformation on the distribution of ions in fibroblasts.
AuthorsLeeves MA, McDonald F
JournalAm J Orthod Dentofacial Orthop
PubMed ID7771368
'The extracellular and intracellular sodium, potassium and chloride concentrations were determined in fibroblast cells located in the rat calvarium. The ionic values were determined by fluorescence microscopy after incubation with the fluorescent probes, sodium-binding benzofuran isophthalate (SBFI), potassium-binding benzofuran isophthalate (PBFI) and 6-methoxy-N-(3-sulfopropyl) quinolinium (SPQ) (Dyes were supplied by CALBIOCHEM, ... More
Inactive fatty acids are unable to flip-flop across the lipid bilayer.
AuthorsJezek P, Modrianský M, Garlid KD
JournalFEBS Lett
PubMed ID9187359
'Free fatty acids (FA) were found which did not acidify liposome interior. This is interpreted as their inability to rapidly flip-flop across the lipid bilayer. However, they were able to partition in lipids as detected directly using HPLC or from the shift of their equilibrium binding to acrylodated intestinal binding ... More
Synthesis and characterization of improved chloride-sensitive fluorescent indicators for biological applications.
AuthorsVerkman AS, Sellers MC, Chao AC, Leung T, Ketcham R
JournalAnal Biochem
PubMed ID2751097
'A class of N-substituted quinoline compounds has been introduced recently for the fluorescence measurement of Cl concentration in biological preparations. The most Cl-sensitive compound was 6-methoxy-N-[3-sulfopropyl] quinolinium with peak excitation and emission wavelengths of 350 and 442 nm and a Stern-Volmer constant for quenching by Cl of 118 M-1. Six ... More
Transport function and regulation of mitochondrial uncoupling proteins 2 and 3.
AuthorsJaburek M, Varecha M, Gimeno RE, Dembski M, Jezek P, Zhang M, Burn P, Tartaglia LA, Garlid KD
JournalJ Biol Chem
PubMed ID10473545
'Uncoupling protein 1 (UCP1) dissipates energy and generates heat by catalyzing back-flux of protons into the mitochondrial matrix, probably by a fatty acid cycling mechanism. If the newly discovered UCP2 and UCP3 function similarly, they will enhance peripheral energy expenditure and are potential molecular targets for the treatment of obesity. ... More
Transport activity of AE3 chloride/bicarbonate anion-exchange proteins and their regulation by intracellular pH.
AuthorsSterling D, Casey JR
JournalBiochem J
PubMed ID10548554
'Plasma membrane Cl(-)/HCO(3)(-) anion-exchange (AE) proteins contribute to regulation of intracellular pH (pH(i)). We characterized the transport activity and regulation by pH(i) of full-length AE3 and the cardiac isoform, AE3c, both of which are expressed in the heart. AE3c is an N-terminal variant of AE3. We also characterized AE1, AE2 ... More
An ATP-driven Cl- pump regulates Cl- concentrations in rat hippocampal neurons.
AuthorsInoue M, Hara M, Zeng XT, Hirose T, Ohnishi S, Yasukura T, Uriu T, Omori K, Minato A, Inagaki C
JournalNeurosci Lett
PubMed ID1667680
'To investigate the role of Cl(-)-stimulated Mg(2+)-ATPase (Cl(-)-ATPase) in neurons, we examined the effects of ethacrynic acid (0.3 mM), which completely inhibits Cl(-)-ATPase on the intracellular Cl- concentrations of cultured rat hippocampal neurons, using Cl(-)-sensitive fluorescent probes. Ethacrynic acid and ATP consuming treatment increased the intracellular Cl- concentration, but elevation ... More
Measuring pore formation by Bcl-2 family proteins.
AuthorsSchendel SL, Reed JC
JournalMethods Enzymol
PubMed ID10914025
'Two methods for assaying Bcl-2 protein family-induced solute efflux from liposomes have been outlined. They utilize either ion-selective electrodes to follow ion efflux or fluorescence to monitor changes in fluorescence of the liposome-encapsulated dye SPQ or carboxyfluorescein. Both methods provide a simple means of determining protein activity. These methods do ... More
Determination of ion permeability by fluorescence quenching.
AuthorsGarcia AM
JournalMethods Enzymol
PubMed ID1382199
Phase fluorometric method for determination of standard lifetimes.
AuthorsThompson RB, Gratton E
JournalAnal Chem
PubMed ID3382018
Optical methods to measure membrane transport processes.
AuthorsVerkman AS
JournalJ Membr Biol
PubMed ID8606370
Chloride fluxes across Acholeplasma laidlawii membranes.
AuthorsSchummer U, Schiefer HG
JournalFEMS Microbiol Lett
PubMed ID1778416
Chloride fluxes across the cytoplasmic membrane of Acholeplasma laidlawii were studied by using the chloride sensitive fluorescent dye, 6-methoxy-N-(sulfopropyl)quinolinium. Chloride was found to penetrate the membrane passively. Chloride flux was dependent upon the transmembrane electric potential. ... More
A microplate fluorimetric assay for measuring dehalogenase activity.
AuthorsMarchesi JR
JournalJ Microbiol Methods
PubMed ID14500026
A fluorimetric assay was developed to measure halide release from halogenated compounds being degraded by microbes. The method relies on the property of halides to quench the fluorescence of 6-methoxy-N-(3-sulfopropyl)quinolinium (SPQ) by collision quenching. The assay shows a wide response to halide concentration (1-500 mM) and tolerates a wide pH ... More
Detection of cell-cycle stage by fluorescence in situ hybridization: its application in human interphase cytogenetics.
AuthorsMukherjee AB, Murty VV, Chaganti RS
JournalCytogenet Cell Genet
PubMed ID1395732
Distinct cell-cycle-dependent changes in the conformation of centromeric chromatin in a specific human chromosome containing alpha-satellite DNA have been demonstrated by fluorescence in situ hybridization (FISH). This method, based upon specific FISH signal morphology, allows simultaneous analysis of chromosomal aneuploidy and detection of specific cell-cycle stage(s) of human tumor and/or ... More
Analysis of image sequences in fluorescence videomicroscopy of stationary objects.
AuthorsBonnet N, Zahm JM
JournalCytometry
PubMed ID9515721
Fluorescence videomicroscopy allows the temporal behavior of biological specimens to be studied at the cellular level. We describe two types of methods that can be used for extracting quantitative information from image sequences: the modeling approach, which is mainly local, and multivariate statistical analysis, which provides a global approach. The ... More
A structure-activity study of fatty acid interaction with mitochondrial uncoupling protein.
AuthorsJezek P, Modrianský M, Garlid KD
JournalFEBS Lett
PubMed ID9187360
Fatty acid (FA) uniport via mitochondrial uncoupling protein (UcP) was detected fluorometrically with PBFI, potassium-binding benzofuran phthalate and SPQ, 6-methoxy-N-(3-sulfopropyl)-quinolinium, indicating K+ and H+, respectively. The FA structural patterns required for FA flip-flop, UcP-mediated FA uniport, activation of UcP-mediated H+ transport in proteoliposomes, and inhibition of UcP-mediated Cl- uniport by ... More
Continuous monitoring of transport by fluorescence on cells and vesicles.
AuthorsEidelman O, Cabantchik ZI
JournalBiochim Biophys Acta
PubMed ID2686755
Fluorescence techniques are gaining wider applicability in the field of membrane transport due to their high temporal resolution, modest demand for biological material and the kinetic information which is made available by fluorescence tracings. The development of novel fluorescent substrates for particular transport systems and of novel fluorescent indicators for ... More
Loading of cells and a possible upper limit of load response with respect to strain energy density.
AuthorsMcDonald F, Yettram AL
JournalJ Biomed Mater Res
PubMed ID8600148
A model has been established to examine the effects of mechanically loading bone on the ionic distribution in osteocytes. The ionic concentrations are determined from the fluorescence of specific ionic indicators SPFI, PBFI, and SPQ. A deformation was induced in the calvariae of neonatal rats which were used as the ... More
A single conductance pore for chloride ions formed by two cystic fibrosis transmembrane conductance regulator molecules.
AuthorsZerhusen B, Zhao J, Xie J, Davis PB, Ma J
JournalJ Biol Chem
PubMed ID10075649
The cystic fibrosis transmembrane conductance regulator (CFTR) is a cAMP-dependent protein kinase (PKA)- and ATP-regulated chloride channel, whose gating process involves intra- or intermolecular interactions among the cytosolic domains of the CFTR protein. Tandem linkage of two CFTR molecules produces a functional chloride channel with properties that are similar to ... More
CFTR chloride channel regulation by an interdomain interaction.
AuthorsNaren AP, Cormet-Boyaka E, Fu J, Villain M, Blalock JE, Quick MW, Kirk KL
JournalScience
PubMed ID10521352
The cystic fibrosis gene encodes a chloride channel, CFTR (cystic fibrosis transmembrane conductance regulator), that regulates salt and water transport across epithelial tissues. Phosphorylation of the cytoplasmic regulatory (R) domain by protein kinase A activates CFTR by an unknown mechanism. The amino-terminal cytoplasmic tail of CFTR was found to control ... More
Uptake of fluorescent dyes associated with the functional expression of the cystic fibrosis transmembrane conductance regulator in epithelial cells.
AuthorsWersto RP, Rosenthal ER, Crystal RG, Spring KR
JournalProc Natl Acad Sci U S A
PubMed ID8577734
Specific mutations in the cystic fibrosis transmembrane conductance regulator (CFTR), the most common autosomal recessive fatal genetic disease of Caucasians, result in the loss of epithelial cell adenosine 3',5'-cyclic-monophosphate (cAMP)-stimulated Cl- conductance. We show that the influx of a fluorescent dye, dihydrorhodamine 6G (dR6G), is increased in cells expressing human ... More
The phorbol ester PMA and cyclic AMP activate different Cl(-) and HCO3(-) fluxes in C127 cells expressing CFTR.
AuthorsZegarra-Moran O, Porcelli AM, Rugolo M
JournalBiochim Biophys Acta
PubMed ID11342000
In mouse mammary epithelial C127 cells expressing wild-type cystic fibrosis transmembrane conductance regulator (CFTR), chloride efflux, measured with the Cl(-)-sensitive dye 6-methoxy-N-(3-sulfopropyl)quinolinium (SPQ), was stimulated by activation of protein kinase A with cyclic AMP elevating agents forskolin plus 3-isobutyl-1-methyl-xanthine (IBMX) and, to a less extent, by activation of protein kinase ... More
Sensitive procedures for measuring chloride fluxes mediated by the purified glycine receptor incorporated into phospholipid vesicles.
AuthorsGarcía-Calvo M, Valdivieso F, Mayor F, Vázquez J
JournalNeurosci Lett
PubMed ID1321962
Two novel methods have been developed to directly measure chloride influx into purified glycine receptor-containing phospholipid vesicles. Using a method based on the fluorescence quenching of a chloride-sensitive probe entrapped into the vesicles, a chloride influx was detected which could be enhanced by glycine and completely abolished by the antagonist ... More
Cell cycle dependence of chloride permeability in normal and cystic fibrosis lymphocytes.
AuthorsBubien JK, Kirk KL, Rado TA, Frizzell RA
JournalScience
PubMed ID2162561
Cystic fibrosis (CF) is a genetic disease characterized by abnormal regulation of epithelial cell chloride channels. Nonepithelial cells, including lymphocytes and fibroblasts, may exhibit a similar defect. Two independent techniques were used to assess the macroscopic chloride permeability (PCl) of freshly isolated B lymphocytes and of B and T lymphocyte ... More
Chloride fluxes activated by parathyroid hormone in human erythrocytes.
AuthorsSoldati L, Adamo D, Spaventa R, Bianchi G, Vezzoli G
JournalBiochem Biophys Res Commun
PubMed ID10708577
We used the chloride fluorescent probe, 6-methoxy-N-(3-sulfopropyl)quinolinium (SPQ), to study chloride fluxes in human erythrocytes. The SPQ load was made by hypotonic buffer (150 mOsm, 10 min). Intracellular fluorescence was monitored continuously at 360 nm excitation and 410 nm emission wavelengths. The leakage of SPQ out of cells was <5% ... More
[Ca2+]i modulation of Cl- content controls cell volume in single salivary acinar cells during fluid secretion.
AuthorsFoskett JK
JournalAm J Physiol
PubMed ID2260645
Differential interference contrast microscopy and low-light-level digital imaging of the fluorescent chloride indicator dye 6-methyl-1-(3-sulfonatopropyl)quinolinium (SPQ) were performed simultaneously in single mammalian salivary gland acinar cells to examine the relationship between cytoplasmic chloride concentration [( Cl-]i) and cell volume during stimulus-secretion coupling. Agonist stimulation of Cl(-)-driven fluid secretion is associated ... More
Phosphorylation of the R domain by cAMP-dependent protein kinase regulates the CFTR chloride channel.
CFTR, the protein associated with cystic fibrosis, is phosphorylated on serine residues in response to cAMP agonists. Serines 660, 737, 795, and 813 were identified as in vivo targets for phosphorylation by protein kinase A. The SPQ fluorescence assay revealed that mutagenesis of any one of these sites did not ... More
Protein kinase A regulates chloride conductance in endocytic vesicles from proximal tubule.
AuthorsBae HR, Verkman AS
JournalNature
PubMed ID1701220
Regulation of ion transport by phosphorylation and G proteins occurs in several epithelial and non-epithelial cell plasma membranes1-5. It is not known whether transporters on intracellular membranes are target sites for second messengers. Here we present direct evidence that a chloride conductance in endocytic vesicles from rabbit proximal tubule is ... More
Membrane chloride transport measured using a chloride-sensitive fluorescent probe.
AuthorsIllsley NP, Verkman AS
JournalBiochemistry
PubMed ID3567167
Transport of chloride across cell membranes through exchange, cotransport, or conductive pathways is a subject of great biological importance. Current methods of measurement are restricted in their sensitivity, time resolution, and applicability. A new transport measurement technique has been developed on the basis of the fluorescence quenching by chloride of ... More
Protein kinase C activates chloride conductance in C127 cells stably expressing the cystic fibrosis gene.
AuthorsDechecchi MC, Tamanini A, Berton G, Cabrini G
JournalJ Biol Chem
PubMed ID7684379
The regulatory domain (R domain) of the cystic fibrosis transmembrane conductance regulator (CFTR) is phosphorylated by protein kinase A and protein kinase C (PKC) in vivo (Picciotto, M. R., Cohn, J. A., Bertuzzi, G., Greengard, P., and Nairn, A. C. (1992) J. Biol. Chem. 267, 12742-12752), but so far the ... More
Cystic fibrosis transmembrane conductance regulator facilitates ATP release by stimulating a separate ATP release channel for autocrine control of cell volume regulation.
AuthorsBraunstein GM, Roman RM, Clancy JP, Kudlow BA, Taylor AL, Shylonsky VG, Jovov B, Peter K, Jilling T, Ismailov II, Benos DJ, Schwiebert LM, Fitz JG, Schwiebert EM
JournalJ Biol Chem
PubMed ID11110786
These studies provide evidence that cystic fibrosis transmembrane conductance regulator (CFTR) potentiates and accelerates regulatory volume decrease (RVD) following hypotonic challenge by an autocrine mechanism involving ATP release and signaling. In wild-type CFTR-expressing cells, CFTR augments constitutive ATP release and enhances ATP release stimulated by hypotonic challenge. CFTR itself does ... More
Intracellular chloride in submucosal gland cells.
AuthorsDwyer TM, Farley JM
JournalLife Sci
PubMed ID2034038
The chloride ion concentration within isolated tracheal submucosal gland cells was studied micro-spectrofluorometrically using a fluorescent dye, 6-methoxy-N-(3-sulfopropyl) quinolinium (SPQ), that is quenched by Cl-. Cells from normal weanling swine and from a cystic fibrosis (CF) patient were used. Ion substitution experiments showed that cell fluorescence increased in both cell ... More
Quenching mechanism of quinolinium-type chloride-sensitive fluorescent indicators.
AuthorsJayaraman S, Verkman AS
JournalBiophys Chem
PubMed ID10885398
Quinolinium based Cl- sensitive fluorescent indicators have been used extensively to measure intracellular Cl- activity. To define their fluorescence quenching mechanism, a series of N-methyl quinolinium derivatives were synthesized, including N-methylquinolinium (Q), 6-methylQ, 6-methoxyQ, 6-chloroQ, 3-bromoQ, 6-aminoQ and N-methylisoquinolinium. Stern-Volmer plots for quenching by Cl-, Br-, SCN-, I-, F-, OAc- ... More
Transport of anions and protons by the mitochondrial uncoupling protein and its regulation by nucleotides and fatty acids. A new look at old hypotheses.
AuthorsJezek P, Orosz DE, Modriansky M, Garlid KD
JournalJ Biol Chem
PubMed ID7929332
The uncoupling protein generates heat by catalyzing electrophoretic proton transport across the inner membrane of brown adipose tissue mitochondria. It also transports Cl- and other monovalent anions, and both proton and anion transport are inhibited by purine nucleotides. Several long-standing hypotheses bear on specific aspects of Cl- transport, H+ transport, ... More
Partial C-terminal unfolding is required for channel formation by staphylococcal alpha-toxin.
AuthorsVécsey-Semjén B, Möllby R, van der Goot FG
JournalJ Biol Chem
PubMed ID8621496
The pore-forming alpha-toxin from Staphylococcus aureus is secreted as a soluble monomeric protein. In order to form a transmembrane channel, the protein has to undergo oligomerization and membrane insertion. Previous studies have shown that channel formation is favored by acidic pH. We have analyzed the effect of pH on the ... More
cAMP-inducible chloride conductance in mouse fibroblast lines stably expressing the human cystic fibrosis transmembrane conductance regulator.
AuthorsRommens JM, Dho S, Bear CE, Kartner N, Kennedy D, Riordan JR, Tsui LC, Foskett JK
JournalProc Natl Acad Sci U S A
PubMed ID1715567
A cAMP-inducible chloride permeability has been detected in mouse fibroblast (L cell) lines upon stable integration of a full-length cDNA encoding the human cystic fibrosis transmembrane conductance regulator (CFTR). As indicated by a Cl(-)-indicator dye, the Cl- permeability of the plasma membrane increases by 10- to 30-fold within 2 min ... More
Reversal of cystic fibrosis phenotype in a cultured Delta508 cystic fibrosis transmembrane conductance regulator cell line by oligonucleotide insertion.
Cystic fibrosis (CF) is a lethal genetic disorder that is due to mutations in the gene encoding the cAMP-activated anion CF transmembrane conductance regulator (CFTR) channel. A three-nucleotide base deletion (TTT), encoding phenylalanine in position 508 of the translatable CFTR sequence (accompanied by a C to T replacement immediately 5' ... More
Conformational changes due to membrane binding and channel formation by staphylococcal alpha-toxin.
AuthorsVécsey-Semjén B, Lesieur C, Möllby R, van der Goot FG
JournalJ Biol Chem
PubMed ID9038182
Conformational changes occurring upon membrane binding and subsequent insertion of staphylococcal alpha-toxin were studied using complementary spectroscopic techniques. Experimental conditions were established where binding could be uncoupled from membrane insertion but insertion and channel formation seemed to be concomitant. Binding led to changes in tertiary structure as witnessed by an ... More
Protein kinase A dependent membrane protein phosphorylation and chloride conductance in endosomal vesicles from kidney cortex.
AuthorsReenstra WW, Sabolic I, Bae HR, Verkman AS
JournalBiochemistry
PubMed ID1310027
Regulation of Cl conductance by protein kinase A may play a role in control of endosomal acidification [Bae, H.-R., & Verkman, A. S. (1990) Nature, 348, 637-639]. To investigate the mechanism of kinase A action, cell-free measurements of Cl transport and membrane protein phosphorylation were carried out in apical endocytic ... More
A single mutation in uncoupling protein of rat brown adipose tissue mitochondria abolishes GDP sensitivity of H+ transport.
The uncoupling protein is one of a family of mitochondrial transport proteins involved in energy metabolism. It dissipates oxidative energy to generate heat, either by catalyzing proton transport directly or by catalyzing fatty acid anion transport, thus enabling fatty acids to act as cycling protonophores. This transport process is tightly ... More
The goal of this study was to develop long-wavelength fluorescent Cl indicators that have improved optical properties over quinolinium compounds. A series of quaternized tricyclic heterocycles was screened. We found that N,N-dimethyl-9,9-bisacridinium (lucigenin) had very high halide sensitivity with Stern-Volmer constants for collisional quenching of 390 M-1 (Cl), 585 M-1 ... More
Evidence against the acidification hypothesis in cystic fibrosis.
AuthorsGibson GA, Hill WG, Weisz OA
JournalAm J Physiol Cell Physiol
PubMed ID11003589
The pleiotropic effects of cystic fibrosis (CF) result from the mislocalization or inactivity of an apical membrane chloride channel, the cystic fibrosis transmembrane conductance regulator (CFTR). CFTR may also modulate intracellular chloride conductances and thus affect organelle pH. To test the role of CFTR in organelle pH regulation, we developed ... More
Regulation of Cl- permeability in normal and cystic fibrosis sweat duct cells.
AuthorsRam SJ, Weaver ML, Kirk KL
JournalAm J Physiol
PubMed ID2240198
Reabsorptive cells of the human sweat gland normally exhibit a high basal Cl- permeability but are markedly impermeable to Cl- in cystic fibrosis (CF). We examined the possibility that the reduced basal Cl- permeability of CF sweat duct cells in primary culture is due to a defective regulation of plasma ... More
Chloride transport in apical membrane vesicles from bovine tracheal epithelium: characterization using a fluorescent indicator.
AuthorsFong P, Illsley NP, Widdicombe JH, Verkman AS
JournalJ Membr Biol
PubMed ID3210223
C1 transport in apical membrane vesicles derived from bovine tracheal epithelial cells was studied using the C1-sensitive fluorescent indicator 6-methoxy-N-(3-sulfopropyl) quinolinium. With an inwardly directed 50 mM C1 gradient at 23 degrees C, the initial rate of C1 entry (JC1) was increased significantly from 0.32 +/- 0.12 nmol.sec-1.mg protein-1 (mean ... More
Chloride currents in primary cultures of rabbit proximal and distal convoluted tubules.
AuthorsRubera I, Tauc M, Bidet M, Poujeol C, Cuiller B, Watrin A, Touret N, Poujeol P
JournalAm J Physiol
PubMed ID9815124
Cl- conductances were studied in cultured rabbit proximal convoluted tubule (PCT) epithelial cells and compared with those measured in cultured distal bright convoluted tubule (DCTb) epithelial cells. Using the whole cell patch-clamp technique, three types of Cl- conductances were identified in DCTb cultured cells. These consisted of volume-sensitive, Ca2+-activated, and ... More
Association of domains within the cystic fibrosis transmembrane conductance regulator.
The cystic fibrosis transmembrane conductance regulator (CFTR) is a Cl- channel composed of two membrane-spanning domains (MSD), two nucleotide-binding domains (NBD), and an R domain. To understand how these domains interact, we expressed various constructs of CFTR containing membrane-spanning and/or cytosolic domains either separately or together. We then tested for ... More
A microplate assay measuring chloride ion channel activity.
AuthorsWest MR, Molloy CR
JournalAnal Biochem
PubMed ID8921165
A microplate chloride ion channel assay, using N-(6-methoxyquinolyl) acetoethyl ester (MQAE) fluorescence changes has been developed. Forskolin stimulation of T84 cells caused cAMP-dependent, increased Cl- loss. Forskolin responses after 6 min gave an EC50 of 0.27 +/- 0.05 microM, illustrating the reproducibility of the assay. Forskolin exposure of CFPAC-1 cells, ... More
Functional reconstitution and characterization of recombinant human alpha 1-glycine receptors.
AuthorsCascio M, Shenkel S, Grodzicki RL, Sigworth FJ, Fox RO
JournalJ Biol Chem
PubMed ID11145968
By utilizing a baculoviral expression system described previously (Cascio, M., Schoppa, N. E., Grodzicki, R. L., Sigworth, F. J., and Fox, R. O. (1993) J. Biol. Chem. 268, 22135-22142), functional recombinant homomeric human alpha(1)-glycine receptors (GlyR) were overexpressed in insect cell culture, solubilized, purified, and reconstituted into lipid vesicles via ... More
Buffer-dependent pH sensitivity of the fluorescent chloride-indicator dye SPQ.
AuthorsVasseur M, Frangne R, Alvarado F
JournalAm J Physiol
PubMed ID8381589
The fluorescence intensity of 6-methoxy-N-(3-sulfopropyl)quinolinium (SPQ) in an N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES) 2-(N-morpholino)ethanesulfonic acid (MES)-Tris(hydroxymethyl)aminomethane buffer, pH 7.0, decreased as a function of Cl- concentration and/or gluconate concentration, as expected. Contrary to expectation, however, the fluorescence intensity progressively increased as the pH decreased, independently of the presence of gluconate. Consequently, the ... More
Fluorescence measurements of anion transport by the GABAA receptor in reconstituted membrane preparations.
AuthorsDunn SM, Shelman RA, Agey MW
JournalBiochemistry
PubMed ID2543444
A fluorescence assay for measuring the functional properties of the GABAA receptor in reconstituted membrane vesicles is described. This assay is based on a method previously described to measure monovalent cation transport mediated by the nicotinic acetylcholine receptor in membranes from Torpedo electric organ [Moore, H.-P.H., & Raftery, M. A. ... More
The human neuroblastoma cell line, IMR-32 possesses a GABAA receptor lacking the benzodiazepine modulatory site.
AuthorsAnderson SM, De Souza RJ, Cross AJ
JournalNeuropharmacology
PubMed ID8391667
GABAA receptors were identified in IMR-32 cell membranes by the binding of [35S]t-butyl-bicyclophosphorothionate ([35S]TBPS) to the chloride channel. GABA (IC50 2.2 microM), muscimol (IC50 0.8 microM), picrotoxin (IC50 1.7 microM), pentobarbitone (IC50 108 microM), etomidate (IC50 53 microM), chlormethiazole (IC50 98 microM) and Ro 5-3663 (IC50 280 microM) all inhibited ... More
NaCl reflection coefficients in proximal tubule apical and basolateral membrane vesicles. Measurement by induced osmosis and solvent drag.
AuthorsPearce D, Verkman AS
JournalBiophys J
PubMed ID2765660
Two independent methods, induced osmosis and solvent drag, were used to determine the reflection coefficients for NaCl (sigma NaCl) in brush border and basolateral membrane vesicles isolated from rabbit proximal tubule. In the induced osmosis method, vesicles loaded with sucrose were subjected to varying inward NaCl gradients in a stopped-flow ... More
Hormonal regulation of Cl transport in polar airway epithelia measured by a fluorescent indicator.
AuthorsVerkman AS, Chao AC, Hartmann T
JournalAm J Physiol
PubMed ID1310213
Cl transport mechanisms in polarized cultures of canine tracheal epithelium were examined using an Ussing-type chamber with independent mucosal and serosal perfusion. Cl activity was monitored continuously from fluorescence of entrapped 6-methoxy-N-(3-sulfopropyl)quinolinium (SPQ). When added to the serosal (but not mucosal) solution, isoproterenol increased Cl fluxes across the apical membrane ... More
Renal brush-border chloride transport mechanisms characterized using a fluorescent indicator.
AuthorsChen PY, Illsley NP, Verkman AS
JournalAm J Physiol
PubMed ID3337241
Cl transport mechanisms in brush-border membrane vesicles (BBMV) isolated from rabbit renal cortex were characterized using the Cl-sensitive fluorescent indicator 6-methoxy-N-[3-sulfopropyl]quinolinium (SPQ). In control experiments using 36Cl, SPQ did not itself alter rates for Cl transport. Cl fluxes (JCl) in nanomoles per second per milligram vesicle protein in response to ... More
On the mechanism of fatty acid-induced proton transport by mitochondrial uncoupling protein.
AuthorsGarlid KD, Orosz DE, Modrianský M, Vassanelli S, Jezek P
JournalJ Biol Chem
PubMed ID8576230
Uncoupling protein mediates electrophoretic transport of protons and anions across the inner membrane of brown adipose tissue mitochondria. The mechanism and site of proton transport, the mechanism by which fatty acids activate proton transport, and the relationship between fatty acids and anion transport are unknown. We used fluorescent probes to ... More
Measurement of changes in cell volume based on fluorescence quenching.
AuthorsSrinivas SP, Bonanno JA
JournalAm J Physiol
PubMed ID9142868
The intracellular fluorescence of 6-methoxy-N-(3-sulfopropyl)-quinolinium (SPQ), a Cl(-) -sensitive fluorescent dye, is quenched by intracellular organic anions and proteins of unknown identity. The concentration of these intracellular quenchers (ICQs), however, is dependent on cell volume. In the absence of Cl-, changes in the observed SPQ fluorescence may therefore reflect changes ... More
Water permeability measurement in living cells and complex tissues.
AuthorsVerkman AS
JournalJ Membr Biol
PubMed ID10630923
The identification of molecular water transporters and the generation of transgenic mice lacking water transporting proteins has created a need for accurate methods to measure water permeability. This review is focused on methodology to characterize water permeability in living cells and complex multicellular tissues. The utility of various parameters defining ... More
Fluorescent chloride indicators to assess the efficacy of CFTR cDNA delivery.
AuthorsMansoura MK, Biwersi J, Ashlock MA, Verkman AS
JournalHum Gene Ther
PubMed ID10223721
Cl(-)-sensitive fluorescent indicators have been used extensively in cell culture systems to measure the Cl(-)-transporting function of the cystic fibrosis transmembrane conductance regulator protein CFTR. These indicators have been used in establishing a surrogate end point to assess the efficacy of CFTR cDNA delivery in human gene therapy trials. The ... More