1,721,160 research outputs found
Potential-sensitive response mechanism of diS-C3-(5) in biological membranes
No full textThe potential-sensitive response mechanism of 3,3′-dipropylthiodicarbocyanine iodide (diS-C3-(5)) was examined based on our previous model of diS-C3-(5) interaction with brush border membrane vesicles (BBMV) in the absence of a membrane potential. The model contained binding (6 msec), reorientation (30 msec), dimerization (<10 nsec), and translocation (1 sec) reaction steps (Cabrini & Verkman, 1986. J. Membrane Biol.90:163-175). Transmembrane potentials (ψ) were induced in BBMV by K+ gradients and valinomycin. Steady-state diS-C3-(5) fluorescence (excitation 622 nm, emission 670 nm) increased linearly with ψ. The reorientation and translocation reaction steps were resolved by the stopped-flow technique as a biexponential decrease in fluorescence following mixture of diS-C3-(5) with BBMV at varying ψ. The fractional amplitude of the faster exponential increased from 0.36 to 0.73 with increasing ψ (-17 to 87 mV); the time constant for the faster exponential (30-35 msec) was independent of ψ. There were single exponential kinetics (0.5-1.5 sec) for diS-C3-(5) fluorescence response to a rapid (<2 msec) change in ψ in the absence of a diS-C3-(5) concentration gradient. These results, and similar findings in placental brush border vesicles, red cell vesicles, and phosphatidylcholine vesicles, support a translocation mechanism for diS-C3-(5) response, where induced membrane potentials drive diS-C3-(5) redistribution between sites at the inner and outer membrane leaflets, with secondary effects on diS-C3-(5) dimerization and solution/membrane partitioning. Fluorescence lifetime and dynamic depolarization measurements showed no significant change in diS-C3-(5) rotational characteristics or in the polarity of the diS-C3-(5) environment with changes in ψ. Based on the experimental results, a mathematical model is developed to explain the quantitative changes in diS-C3-(5) fluorescence which accompany changes in ψ at arbitrary dye/lipid ratios. © 1986 Springer-Verlag New York Inc
INTERACTION MECHANISM AND LOCALIZATION OF POTENTIAL SENSITIVE CYANINE DYE BINDING TO BRUSH-BORDER MEMBRANES
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Localization of cyanine dye binding to brush-border membranes by quenching of n-(9-anthroyloxy) fatty acid probes
No full textThe location and orientation of 3,3′-dipropylthiodicarbocyanine (diS-C3-(5)) binding sites in renal brush-border membrane vesicles was examined from the quenching of n-(9-anthroyloxy) fatty acid (n-AS) fluorescence. Based on previous kinetic studies (Cabrini, G. and Verkman, A.S. (1986) J. Membrane Biol. 90, 163-175) monomeric aqueous diS-C3-(5) binds to brush-border membrane vesicles (BBMV) by an initial 6 ms association to form bound monomer, a 30-40 ms equilibrium between bound monomer (M) and bound dimer (D), and a 1-1.3 s translocation of D from the outer to inner membrane leaflet. Based on Stern-Volmer and lifetime analyses, M and D quench the fluorescence of the n-AS probes by a collisional mechanism. At low [diS-C3-(5)]/[BBMV] (R), where M predominates, the n-AS quenching efficiencies (Q) are similar (n = 2-16); at high R, where D predominates, Q increases with n (16 > 12 ⋙ 6 > 2), suggesting that M is oriented parallel, and D perpendicular, to the phospholipid chains deep within the membrane. Mixture of diS-C3-(5) with brush-border membrane vesicles containing n-AS in a stopped-flow apparatus gave a biexponential fluorescence decrease (excitation 390 nm, emission above 450 nm) with time constants 30-40 ms and 1-1.5 s; there was no 6 ms quenching process. These findings are incorporated into a model in which diS-C3-(5) adheres loosely to the outer membrane surface in 6 ms, binds parallel to the membrane phospholipid in 30-40 ms, dimerizes and rotates by 90° in much less than 30 ms, and translocates to the opposite half of the bilayer in 1-1.5 s. © 1986
OPTICAL MEASUREMENT OF MEMBRANE-POTENTIAL IN RENAL MEMBRANES - DIS-C3-(5) RESPONSE MECHANISM
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Mechanism of interaction of the cyanine dye DiS-C3-(5) with renal brush-border vesicles
No full textThe equilibrium binding mechanism and kinetics of binding of diS-C3-(5) (3,3′-dipropylthiodicarbocyanine iodide) to rabbit renal brush-border membrane vesicles (BBMV) were examined using steady-state and time-resolved fluorescence, and fluorescence stopped-flow methods. In aqueous solution, diS-C3-(5) exists as a monomer at concentrations <5 μm with fluorescence emission peak at 670 nm (excitation 622 nm), anisotropy r=0.102, and lifetime τ=1.2 nsec (23°C). Upon addition of increasing BBMV (voltage clamped to 0 mV using K+/valinomycin), the 670 nm emission peak decreases, corresponding to formation of a nonfluorescent membrane dimer, and subsequently a new emission peak at 695 nm increases, corresponding to membrane monomer. Dynamic depolarization studies show that aqueous diS-C3-(5) rotation is unhindered with a rotational rate R=0.57 nsec-1 while membrane monomer is hindered with steady-state anisotropy r=0.190, lifetime τ=2.1 nsec, R=0.58 nsec-1 and limiting anisotropy r∝=0.11. Based on equilibrium fluorescence titrations, the membrane monomer-dimer (M-D) dissociation constant, Kd=[M]2/[D][BBMV], is 0.0013, where BBMV is expressed as membrane phospholipid concentration. Three distinct kinetic processes are identified by stopped-flow experiments in which BBMV are mixed with diS-C3-(5). There is rapid binding of diS-C3-(5) to the membrane to form bound monomer with a 6-msec exponential time constant. The membrane monomer at the membrane outer surface then aggregates to form bound dimer at the outer surface with a concentration independent time constant of 30 msec. The overall dimerization reaction probably consists of a rate-limiting reorientation process (30 msec) followed by a rapid dimerization which occurs on a nanosecond time scale. Finally, there is a 0.8 to 1 sec translocation of membrane dimer between symmetric sites at the inner and outer membrane surfaces. The translocation reaction is the step which is probably sensitive to changes in transmembrane electrical potential. © 1986 Springer-Verlag New York Inc
External anions regulate stilbene-sensitive proton transport in placental brush border vesicles
No full textThe mechanism for HCO3 --independent proton permeability in microvillus membrane vesicles (MVV) isolated from human placenta was examined by using the entrapped pH indicator 6-carboxyfluorescein (6CF). Proton fluxes (JH) across MVV were determined in response to induced pH and anion gradients from the time course of 6CF fluorescence, the MVV buffer capacity, and the 6CF vs. pH calibration. In the absence of anions, JH was 12 ± 2 nequiv s-1 (mg of protein)-1 (pHin 7.4, pHout 6.0, MVV voltage-clamped with K+/valinomycin, 23°C), corresponding to a proton permeability coefficient of 0.02 cm/s, with an activation energy of 9.1 ± 0.3 kcal/mol. JH was inhibited 20% by dihydro-4,4′-diisothiocyano-2,2′-stilbenedisulfonic acid (H2DIDS) with KI = 8 μM ([Cl-]out = 0 mM). For a 0.5-unit pH gradient JH increased from 1.5 to 4.6 nequiv s-1 (mg of protein)-1 as the internal MVV pH was increased (5.5-7.5). External Cl-, Br-, and I- (but not SO4 2- and PO4 -) increased JH 1.3-2.5-fold for both inwardly and outwardly directed pH gradients with KD = 1.0 ± 0.4 mM (Br-) and > 100 mM (Cl-). This increase was blocked by 100 μM H2DIDS but not by amiloride or furosemide. Internal Cl- did not alter JH induced by pH gradients nor were proton fluxes induced by anion gradients in the absence of a pH gradient. Experiments in which JH was driven by membrane potentials (induced by valinomycin and K+ gradients) indicated that proton transport was voltage-sensitive. These experiments demonstrate a stilbene-sensitive electrogenic proton transport mechanism in MVV that is regulated allosterically by anions at an external binding site. © 1986 American Chemical Society
Expression, functional analysis, and in situ hybridization of a cloned rat kidney collecting duct water channel.
No full textThe cloning and expression of an apical membrane water channel from rat kidney collecting duct (WCH-CD) homologous to a 28-kDa integral membrane protein (CHIP28) was reported recently (K. Fushimi, S. Uchida, Y. Hara, Y. Hirata, F. Marumo, and S. Sasaki. Nature Lond. 361: 549-552, 1993). We obtained an approximately 1.8-kilobase clone from a rat kidney lambda gt10 cDNA library by a polymerase chain reaction cloning method; whereas the coding sequence (814 base pairs, predicted protein size 29 kDa) was identical to that reported, we identified an in-frame ATG codon at base pair -123 predicting a protein size of 33 kDa. On Northern blots probed by cDNAs corresponding to the WCH-CD coding sequence (base pairs +1 to +814) or 5'-untranslated sequence (-403 to -16), a single band at 1.9 kilobases was observed in kidney medulla greater than in cortex but not in other tissues; mRNA expression was increased strongly by dehydration. Translation and oocyte expression studies were performed to identify the translation start site. The short (base pairs +1 to +814) and long (base pairs -123 to +814) cDNAs were subcloned in vector pSP64 containing the 5'-untranslated Xenopus globin sequence upstream to the ATGs; a 30-base pair c-myc sequence was engineered at the COOH- terminal for antibody recognition.(ABSTRACT TRUNCATED AT 250 WORDS
Transepithelial water permeability in microperfused distal airways. Evidence for channel-mediated water transport
No full textWater movement across the airway epithelium is important for regulation of the volume and composition of airspace fluid. A novel approach is reported here to measure osmotic and diffusional water permeability in intact airways. Small airways (100-200 mu m diameter, 1-2 mm length) from guinea pig lung were microdissected and perfused in vitro using concentric glass holding and perfusion pipettes. For measurement of osmotic water permeability (P-f), the airway lumen was perfused with PBS (300 mOsM) containing a membrane impermeable fluorophore, fluorescein sulfonate (FS), and the airway was bathed in solutions of specified osmolalities, P-f determination was based on the change in FS fluorescence at the distal end of the airway resulting from transepithelial water transport, P-f was 4-5 X 10(-3) cm/s at 23 degrees C and independent of lumen flow rate (10-100 nl/min) and the magnitude and direction of the osmotic gradient (bath osmolality 50-600 mOsM). Temperature dependence measurements gave an activation energy of 4.4 kcal/mol (15-37 degrees C). P-f was not altered by 0.3 mM HgCl2 or 50 mu M forskolin, but was increased to 31 X 10(-3) cm/s by 100 mu g/ml amphotericin B, indicating that osmosis is not limited by unstirred layers. Diffusional water permeability (P-d) was measured by H2O/(DO)-O-2 (deuterium oxide) exchange using the H2O/D2O-sensitive fluorescent probe aminonapthelane trisulfonic acid in the lumen. Measured P-d was 3-6 X 10(-6) cm/s at 23 degrees C, indicating significant restriction to water diffusion by unstirred layers. Antibody localization of water channels showed strong expression of the mercurial-insensitive water channel (AQP-4) at the basolateral membrane of airway epithelial cells. These results provide functional evidence that water movement across the distal airway epithelium is mediated by water channel
Localization and functional analysis of CHIP28k water channels in stably transfected Chinese hamster ovary cells.
No full textCHIP28 is a major water transporting protein in erythrocytes and plasma membranes in kidney proximal tubule and thin descending limb of Henle. Chinese hamster ovary cells were stably transfected with the coding sequence of cloned rat kidney CHIP28k using expression vectors containing cytomegalovirus or Rous sarcoma virus promoters. Clonal cell populations expressed a 1.3-kilobase mRNA on Northern blot probed by CHIP28k cDNA and a 28-kDa protein on immunoblot probed by a polyclonal CHIP28 antibody. The clone with greatest expression produced approximately 8 x 10(6) copies of CHIP28k protein/cell. Plasma membrane osmotic water permeability (Pf), measured by stopped-flow light scattering, was 0.004 cm/s in control (vector-transfected) cells (10 degrees C) and 0.014 cm/s in the CHIP28k-transfected cells. Pf in CHIP28k-transfected cells had an activation energy of 4.9 kcal/mol and was reversibly inhibited by HgCl2. CHIP28k expression did not affect the transport of protons and the small polar non-electrolytes urea and formamide. CHIP28k immunoreactivity and function was then determined in subcellular fractions. Pf in 6-carboxyfluorescein-labeled endocytic vesicles, measured by a stopped-flow fluorescence quenching assay, was 0.002 cm/s (control cells) and 0.011 cm/s (CHIP28k-transfected cells); Pf in transfected cells was inhibited by HgCl2. Immunoblotting of fractionated endoplasmic reticulum, Golgi, and plasma membranes revealed high densities of CHIP28k (approximately 5000 monomers/microns 2 in plasma membrane) with different glycosylation patterns; functional water transport activity was present only in Golgi and plasma membrane vesicles. Antibody detection of CHIP28k by confocal fluorescence microscopy and immunogold electron microscopy revealed localization to plasma membrane and intracellular vesicles. These studies establish a stably transfected somatic cell line that strongly expresses functional CHIP28k water channels. As in the original proximal tubule cells, the expressed CHIP28k protein is a selective water channel that is functional in endocytic vesicles and the cell plasma membrane
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