valinomycin and potassium-thiocyanate

The cystic fibrosis transmembrane conductance regulator (CFTR) contains two membrane-spanning domains; each consists of six transmembrane segments joined by three extracellular and two intracellular loops of different length. To examine the role of intracellular loops in CFTR channel function, we studied a deletion mutant of CFTR (delta 19 CFTR) in which 19 amino acids were removed from the intracellular loop joining transmembrane segments IV and V. This mutant protein was expressed in a human embryonic kidney cell line (293 HEK). Fully mature glycosylated CFTR (approximately 170 kDa) was immunoprecipitated from cells transfected with wild-type CFTR cDNA, while cells transfected with the mutant gene expressed only a core-glycosylated form (approximately 140 kDa). The chloride efflux rate (measured by 6-methoxyl-N-(3-sulfopropyl) quinolinium SPQ fluorescence) from cells expressing wild-type CFTR increased 600% in response to forskolin. In contrast, delta 19 CFTR-expressing cells had no significant response to forskolin. Western blotting performed on subcellular membrane fractions showed that delta 19 CFTR was located in the same fractions as delta F508 CFTR, a processing mutant of CFTR. These results suggest that delta 19 CFTR is located in the intracellular membranes, without reaching the cell surface. Upon reconstitution into lipid bilayer membranes, delta 19 CFTR formed a functional Cl- channel with gating properties nearly identical to those of the wild-type CFTR channel. However, delta 19 CFTR channels exhibited frequent transitions to a 6-picosiemens subconductance state, whereas wild-type CFTR channels rarely exist in this subconductance state. These data suggest that the intracellular loop is involved in stabilizing the full conductance state of the CFTR Cl- channel.

Topics: Amino Acid Sequence; Blotting, Western; Cell Line; Chlorides; Colforsin; Cystic Fibrosis Transmembrane Conductance Regulator; Embryo, Mammalian; Humans; Ion Channel Gating; Kidney; Kinetics; Lipid Bilayers; Membrane Potentials; Molecular Sequence Data; Mutagenesis; Protein Structure, Secondary; Recombinant Proteins; Sequence Deletion; Thiocyanates; Valinomycin

1. Activated delta-endotoxins from Bacillus thuringiensis strains toxic to lepidopteran larvae inhibit K(+)-dependent accumulation of amino acids into brush border membrane vesicles (BBMV) from the midgut of the susceptible species Bombyx mori. 2. The activated toxins interfere with the K(+)-dependent uptake of histidine into BBMV only if they are effectively active in vivo. 3. To calculate IC50 values (the toxin concentration which determines 50% of the effect), dose-response curves were performed for each toxin. The values obtained correlate well with the LD50 determined by bioassay. 4. This amino acid inhibition test could represent a rapid (3-6 hr, compared to 3-4 days for bioassay) and sensitive method for the screening of larvicidal activity of known or new recombinant delta-endotoxins.

Topics: Animals; Bacillus thuringiensis; Bacillus thuringiensis Toxins; Bacterial Proteins; Bacterial Toxins; Biological Assay; Bombyx; Endotoxins; Hemolysin Proteins; Histidine; Larva; Lethal Dose 50; Microvilli; Potassium; Thiocyanates; Valinomycin

Transport of glutamine across the brush-border membrane of the rat intestine was examined using brush-border membrane vesicle (BBMV) technique. Osmolarity and temperature studies indicated that the uptake of glutamine by BBMV is mostly the result of transport of the substrate into the intravesicular space. Transport of glutamine was Na+-gradient dependent (out greater than in) with a distinct 'overshoot' phenomenon. Initial rate of transport of glutamine as a function of concentration was saturable both in the presence and absence of a Na+ gradient (out greater than in). Apparent Km of 3.50 and 3.34 mM and Vmax of 707 and 282 pmol/mg protein per 7 s, were calculated for the Na+-dependent and the Na+-independent transport processes of glutamine. The transport of [3H]glutamine by the Na+-dependent and the Na+-independent processes was severely inhibited by the addition to the incubation medium of other amino acids and unlabelled glutamine. Inducing a relatively negative intravesicular compartment with the use of valinomycin and an outwardly directed K+ gradient stimulated glutamine transport. This indicates that transport of the substrate by the Na+-dependent process is electrogenic in nature. Transport of glutamine by the Na+-independent process, however, appeared to be electroneutral in nature. These results demonstrate the existence of two carrier-mediated transport processes for glutamine in the rat intestinal BBMV, one is Na+-dependent and the other is Na+-independent. Furthermore, the results suggest that glutamine transport by the Na+-dependent process probably occurs by a glutamine/Na+ cotransport mechanism.

Topics: Animals; Asparagine; Biological Transport; Electrochemistry; Glutamine; Intestinal Mucosa; Kinetics; Male; Membrane Potentials; Microvilli; Osmolar Concentration; Potassium; Potassium Chloride; Rats; Rats, Inbred Strains; Serine; Sodium; Thiocyanates; Valinomycin

Addition of A23187 plus EDTA to energized mitochondria in KCl medium determines a rapid osmotic swelling due to K+ uptake. The swelling is fully reversed by uncoupler, is stimulated by quinine, and is accompanied by membrane depolarization and increased rate of respiration. A23187-treated mitochondria passively swell in K+ thiocyanate at neutral pH, under conditions where the H+-K+ antiporter appears to be silent. These data indicate that A23187 activates electrophoretic K+ flux, supporting the notion that Mg2+ depletion unmasks several ionic conductance pathways whose concerted interplay could provide a sensitive regulation of mitochondrial volume homeostasis.

Topics: Calcimycin; Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone; Intracellular Membranes; Kinetics; Membrane Potentials; Mitochondria; Mitochondrial Swelling; Potassium; Potassium Chloride; Quinine; Thiocyanates; Valinomycin

Cell suspensions of acetate-grown Methanosarcina barkeri mediate the conversion of CO and H2O to CO2 and H2. The reaction is coupled with the phosphorylation of ADP. Evidence is presented that CO oxidation by the cells is associated with the transient acidification of the suspension medium. Up to 2 mol vectorial protons were measured/mol CO oxidized when the transmembrane electrical gradient was kept low by the addition of valinomycin (20 nmol/mg protein) and KCl (200 mM) or of KSCN (50 mM). No transient acidification was observed in the presence of the protonophore tetrachlorosalicylanilide which stimulated rather than inhibited CO oxidation. Proton extrusion remained unaltered when the proton-translocating ATPase was specifically inhibited by dicyclohexylcarbodiimide. The latter finding indicates that proton translocation is associated with CO conversion to CO2 and H2 rather than with ATP hydrolysis in the cells. The data substantiate that the coupling of CO oxidation with ADP phosphorylation in M. barkeri occurs via a chemiosmotic mechanism.

Topics: Archaea; Bacteria; Biological Transport; Carbon Dioxide; Carbon Monoxide; Dicyclohexylcarbodiimide; Hydrogen-Ion Concentration; Hydrolysis; Membrane Potentials; Oxidation-Reduction; Potassium Chloride; Protons; Thiocyanates; Valinomycin

The role of the transmembrane potential (delta phi), the proton concentration gradient (delta pH) and the proton electrochemical gradient (delta gamma H+) in monoamine uptake by bovine chromaffin granules or ghosts was investigated. In presence of ATP the permeant anion SCN- collapsed the delta phi (inside positive) and inhibited monoamine uptake by granules or well buffered ghosts. With lightly buffered ghosts, SCN- induced an acidification which resulted in a low inhibition of uptake. Cation efflux as well as anion influx affected the delta phi, and a transient valinomycin-mediated K+ efflux induced a lag in the uptake. The delta pH-driven noradrenalin uptake was also sensitive to delta phi, since superimposing a positive or a negative delta phi to the delta pH, respectively, increased or decreased the rate of noradrenalin accumulation. A delta pH was required for this increase of uptake rate, adrenalin accumulation. A delta pH was required for this increase of uptake rate, which was proportional to the delta phi. The pH-dependence of the ATP-induced monoamine uptake by granules pointed to the delta gamma H+ as the driving force. In contrast with the rate of uptake, which was not dependent on the anions present, the extent of amine incorporation was decreased when the internal anionic buffer concentration was decreased and, at a low internal buffer concentration, when ATP anion transport was blocked.

Topics: Adenosine Triphosphate; Animals; Biological Transport, Active; Cattle; Chromaffin Granules; Chromaffin System; Hydrogen-Ion Concentration; Membrane Potentials; Methylamines; Norepinephrine; Potassium; Thiocyanates; Tyramine; Valinomycin