Membrane potentials associated with Ca-induced K conductance in human red blood cells: Studies with a fluorescent oxonol dye,WW 781

Summary A divalent anionic dye, bis-[3-methyl-1-p-sulfophenyl-5-pyrazolone-(4)]-pentamethine oxonol (WW 781) is a rapidly responding fluorescent indicator of KCl diffusion potentials induced in human red blood cells with valinomycin, gramicidin, and with the Ca ionophore A 23187 in the presence of external Ca. WW 781 has a sensitivity of 0.13% F/mV, a detection limit of 10 mV, a response time of less than 1 sec, and exhibits a decrease in fluorescence intensity upon hyperpolarization without detectable shifts in absorption or emission peaks. This dye does not perturb the normal resting potential, and unlike the slow permeant cyanine dyes, does not inhibit Ca-induced K conductance in human red blood cells. However, WW 781 does stimulate Ca-induced unidirectional Rb efflux. With Ca plus A 23187, the initial rapid change in dye fluorescence is sensitive to [Ca] _o and to [A 23187], is reversible with excess EGTA, and is inhibited by quinine, oligomycin, and by trifluoperazine. A biphasic dependence of hyperpolarization on K _o is evident at pH 6, where the ionic selectivity of activation is K, Rb>Cs>Na and that of conductance is K, Rb>Cs. Conditions were defined which permitted continuous monitoring ofE _m for at least 10 min, and the time dependence of the Ca-induced potentials was characterized. Since the properties of the Ca-induced changes in dye fluorescence correlate well with the known characteristics of Ca-induced K permeability, we conclude that WW 781 is a useful indicator of changes inE _m, provided that sufficient controls are employed to separate direct effects of Ca on dye fluorescence from the effects ofE _m on fluorescence.     

Complexation of polymethine dyes with human serum albumin: a spectroscopic study

Non-covalent interactions between polymethine dyes of various types (cationic and anionic thiacarbocyanines as well as anionic oxonols and tetracyanopolymethines) and human serum albumin (HSA) were studied by means of absorption, fluorescence and circular dichroism (CD) spectroscopies. Complexation with the protein leads to a red shift of the dye absorption spectra and, in most cases, to a growth of the fluorescence quantum yield (Phif; for oxonols this growth is very small). The binding constants (K) obtained from changing the absorption spectra and Phif vary from 10(4) to (5-6) x 10(7) M(-1). K for the anionic dyes is much higher than for the cationic dyes (the highest K was found for oxonols). Interaction of meso-substituted anionic thiacarbocyanines with HSA results in cis-->trans isomerization and, as a consequence, an appearance and a steep rise of dye fluorescence. Binding to HSA gives rise to dye CD signals and in many cases is accompanied by aggregation of the dyes. These aggregates often exhibit biphasic CD spectra. The aggregates formed by the dyes alone are decomposed in the presence of HSA.     

The fluorescent cationic dye rhodamine 6G as a probe for membrane potential in bovine aortic endothelial cells.

The membrane potential of cultured bovine aortic endothelial cells was assessed by a fluorescent probe as an alternative to direct methods. We used the fluorescent cationic dye rhodamine 6G, a lipophilic probe with high permeability in cell membranes. A linear relationship was obtained between fluorescence intensity (F.I.) and membrane potential (Em) as a function of the extracellular Na(+) concentration in the presence of the ionophore gramicidin. From the equation derived from the linear relationship F.I. = -0.004 Em + 0. 03 (P < 0.001), the fluorescence measurements could be converted to membrane potential. The resting plasma membrane potential obtained was -65 +/- 7 mV. Nigericin (27 microM), ouabain (1 mM), and bradykinin (20 nM) induced a decrease in F.I. (depolarization), while ATP (25-100 microM) induced an increase in F.I. (hyperpolarization). Mitochondrial membrane potential inhibitors myxothiazol (3 microM) and oligomycin (4 microM) did not influence F. I. measured in the cultured bovine aortic endothelial cells. The results indicate that rhodamine 6G can be used as a sensitive and specific dye in studies of substances that affect the membrane potential of endothelial cells.     

Depolarization of synaptosomal membranes: a study of mechanism by which rhodamine 6G measures membrane potential

The fluorescence intensity of Rhodamine 6G in synaptosomal suspensions has been measured to monitor the membrane potential changes in pre-synaptic nerve terminals. The fluorescence response of the dye was seen to be a function of potential-dependent partitioning of dye molecules between the synaptosomes and the extracellular medium. Binding of dye molecules to the hydrophobic regions of membranes results in the quenching of fluorescence. Upon depolarization of the synaptosomal membrane, the dye molecules are released from the cells. The effect of changing extracellular ionic composition was also studied. The membrane potential increased linearly with log of [K]0. The resting membrane potential in buffer containing 5 mM K+ was calculated to be -60 mV. Raising the extracellular Ca2+ and Mg2+ from 1.2 mM to 10 mM did not change the membrane potential. Ca2+ ionophore A23187, in the presence of Ca2+ was found to depolarize the membranes.     

Differentiation Between Alterations in Plasma and Mitochondrial Membrane Potentials in Synaptosomes Using a Carbocyanine Dye

The cationic potentiometric fluorescent probe 3,3'-diethylthiadicarbocyanine iodide [DiS-C2(5)] was used in synaptosomes to assess the relative contributions of plasma and mitochondrial membrane potentials (psi p and psi m, respectively) to overall fluorescence. Addition of synaptosomes to media containing 0.5 microM dye caused a decrease in fluorescence intensity due to dye accumulation, which equilibrated usually within 5 min. Depolarization of mitochondria by combined treatment with cyanide and oligomycin increased fluorescence by 42%, indicating significant prior accumulation of dye into intrasynaptosomal mitochondria. psi p was calculated to be -54 mV and was not altered significantly by prior depolarization of psi m with cyanide and oligomycin (hereafter referred to as "poisoned" synaptosomes). Similarly, the linear relationship between dye fluorescence and psi p was not altered by depolarization of psi m. Valinomycin, a K+ ionophore, caused a psi p-dependent increase in fluorescence in control (nonpoisoned) synaptosomes, but did not alter fluorescence of poisoned synaptosomes except when the extracellular concentration of K+ ([K+]e) was 2 mM, in which case valinomycin hyperpolarized psi p by about 5 mV. The pore-forming antibiotic gramicidin depolarized both psi p and psi m maximally. Under these conditions, Triton X-100 further increased fluorescence by 40%, indicating significant dye binding to synaptosomal components. In poisoned synaptosomes depolarized by 75 mM K+, gramicidin caused a decrease in fluorescence intensity (hyperpolarization of psi p). The organic solvent dimethyl sulfoxide, used as a vehicle for the hydrophobic ionophores, had voltage-dependent effects on psi p and psi m.(ABSTRACT TRUNCATED AT 250 WORDS)     

The use of fluorescent dyes to measure membrane potentials: a response

The use of fluorescent cyanine dyes to estimate membrane potential in cell suspensions has been considered. Several problems related tot he application of the dyes have been reviewed. These problems include: 1) alteration of the membrane potential (Em) and factors involved in establishing Em by the dyes themselves, 2) the effects of altered energy metabolism on the fluorescent response of the dyes and on Em, and 3) calibration of dye fluorescence. Recent reports that advocate the use of the fluorescent dyes are misleading.     

Voltage dependence of DIDS-insensitive chloride conductance in human red blood cells treated with valinomycin or gramicidin

Net K and Cl effluxes induced by valinomycin or by gramicidin have been determined directly at varied external K, denoted by [K]o, in the presence and absence of the anion transport inhibitors DIDS (4,4'-diiso- thiocyano-2,2'-disulfonic acid stilbene), and its less potent analogue SITS (4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid). The results confirm that pretreatment with 10 microM DIDS, or 100 microM SITS, for 30 min at 23 degrees C inhibits conductive Cl efflux, measured in the continued presence of the inhibitors at 1 mM [K]o, by only 59-67%. This partial inhibition by 10 microM DIDS at 1 mM [K]o remains constant when the concentration of DIDS, or when the temperature or pH during pretreatment with DIDS, are increased. Observations of such partial inhibition previously prompted the postulation of two Cl conductance pathways in human red blood cells: a DIDS-sensitive pathway mediated by capnophorin (band 3 protein), and a DIDS-insensitive pathway. The present experiments demonstrate that at [K]o corresponding to values of EK between -35 and 0 mV the DIDS- insensitive component of net Cl efflux is negligible, being < or = 0.1 muMol/g Hb/min, both with valinomycin (1 microM) and with gramicidin (0.06 microgram/ml). At lower [K]o, where EK is below approximately -35 mV, the DIDS-insensitive fraction of net Cl efflux increases to 2.6 muMol/g Hb/min with valinomycin (1 microM), and to 4.8 muMol/g Hb/min with gramicidin (0.06 microgram/ml). With net fluxes determined from changes in mean cell volume, and with membrane potentials measured from changes in the external pH of unbuffered red cell suspensions, a current-voltage curve for DIDS-insensitive Cl conductance has been deduced. While specific effects of varied [K]o on net Cl efflux are unlikely but cannot strictly be ruled out, the results are consistent with the hypothesis that DIDS-insensitive Cl conductance turns on at an Em of approximately -40 mV.     

Species specificity in the extrinsic absorption changes exhibited by some invertebrates stained with voltage-sensitive dyes

Summary The absorption changes of several invertebrate neuronal preparations stained by the potentiometric dyes (WW 375, WW 433, WW 401 and RGA 84) in response to electrical nerve stimulation were examined. The dyes did not penetrate the connective sheath of insect preparations, but stained it. Only a decremental spreading of optical signals was seen onPeriplaneta americana, Gryllus bimaculatus and GitGryllus campestris ganglia and nerves. In contrast to insect preparations, pond snail and leech neurons were well stained by these dyes. The dye WW 375 behaved somewhat distinctly on insect and pond snail preparations than had been previously reported on other invertebrates. Like the signals from vertebrate neurons, they usually had triphasic action spectra. Therefore, this kind of action spectrum is not found only in membranes of vertebrate neurons. The main conclusion of this work is that the species-specific effects of the dye on different invertebrate preparations have a common feature: the existence of three peaks in the change of absorption (at 575, 675 and 750 nm) in both kinds of WW 375 action spectra (monophasic or triphasic). The wavelength dependence of the change in absorption was not affected by concentration, staining time, pH, osmolarity or ionic composition of physiological saline.     

Impermeant potential-sensitive oxonol dyes: I. Evidence for an “On-off” mechanism

Summary This series of papers addresses the mechanism by which certain impermeant oxonol dyes respond to membranepotential changes, denoted E _m . Hemispherical oxidized cholesterol bilayer membranes provided a controlled model membrane system for determining the dependence of the light absorption signal from the dye on parameters such as the wavelength and polarization of the light illuminating the membrane, the structure of the dye, and E _m . This paper is concerned with the determination and analysis of absorption spectral changes of the dye RGA461 during trains of step changes ofE _m . The wavelength dependence of the absorption signal is consistent with an on-off mechanism in which dye molecules are driven by potential changes between an aqueous region just off the membrane and a relatively nonpolar binding site on the membrane. Polarization data indicate that dye molecules in the membrane site tend to orient with the long axis of the chromophore perpendicular to the surface of the membrane. Experiments with hyperpolarized human red blood cells confirmed that the impermeant oxonols undergo a potential-dependent partition between the membrane and the bathing medium.     

Spectrophotometric measurements of transmembrane potential and pH gradients in chromaffin granules

The electrical potential (delta psi) and proton gradient (alpha pH) across the membranes of isolated bovine chromaffin granules and ghosts were simultaneously and quantitatively measured by using the membrane- permeable dyes 3,3'dipropyl-2,2'thiadicarbocyanine (diS-C3-(5)) to measure delta psi and 9-aminoacridine or atebrin to measure delta pH. Increases or decreases in the delta psi across the granular membrane could be monitored by fluorescence or transmittance changes of diS-C3- (5). Calibration of the delta psi was achieved by utilization of the endogenous K+ and H+ gradients, and valinomycin or carbonyl cyanide-p- trifluoromethoxyphenylhydrazone (FCCP), respectively, with the optical response of diS-C3-(5) varying linearly with the Nernst potential for H+ and K+ over the range -60 to +90 mV. The addition of chromaffin granules to a medium including 9-aminoacridine or atebrin resulted in a rapid quenching of the dye fluorescence, which could be reversed by agents known to cause collapse of pH gradients. From the magnitude of the quenching and the intragranular water space, it was possible to calculate the magnitude of the alpha pH across the chromaffin granule membrane. The time-course of the potential-dependent transmittance response of diS-C3-(5) and the delta pH-dependent fluorescence of the acridine dyes were studied simultaneously and quantitatively by using intact and ghost granules under a wide variety of experimental conditions. These results suggest that membrane-permeable dyes provide an accurate method for the kinetic measurement of delta pH and delta psi in an amine containing subcellular organelle.     

Kinetics of the association of potential-sensitive dyes with model and energy-transducing membranes: Implications for fast probe response times

Summary The second-order rate constants characterizing the association of potential-sensing dyes of the cyanine, merocyanine, and oxonol classes with glycerylmonooleate suspensions, azolectin vesicles, or submitochondrial particles have been measured and the implications for redistribution type mechanisms proposed to explain the potential-dependent optical signals of these probes considered. The second-order rate constants obtained for the cyanines and oxonols are compatible with microsecond probe response times only on the assumption that a high local dye concentration exists in the aqueous phase immediately adjacent to the membrane surface. Calculations based on a surface charge density induced by a bias potential suggest that the necessary local concentration cannot be attained by a diffusion polarization mechanism. A model based on the rapid recombination of ejected dye with the membrane bilayer seems capable of explaining microsecond probe response times in systems where the potential is rapidly changing polarity; calculations suggest that an ejected dye molecule would not diffuse out of an unstirred layer of 100 microns thickness on a millisecond time scale. Microsecond probe responses are also compatible with a first-order potential-dependent dye ejection from the membrane with no rapid recombination when the potential is not changing polarity. The apparent first-order rate constants describing the interaction of merocanine M-540 with a glycerylmonooleate suspension are independent of dye concentration; the reaction may be diffusion limited. The high local dye concentration need not be met in this case for a mechanism based on the transfer of dye onto the membrane from the aqueous phase to describe the microsecond signals of this dye, but other mechanisms have been proposed to explain such signals. The mechanism leading to potentialdependent signals from optical probes appear to differ substantially between suspensions of energy-transducing biological membranes and those involving excitable membranes such as the squid giant axon or model black lipid membranes.     

Dichroic behavior of the absorbance signals from dyes NK2367 and WW375 in skeletal muscle fibers

Absorbance signals were recorded from voltage-clamped single muscle fibers stained with the nonpenetrating potentiometric dyes NK2367 and WW375 and illuminated with quasimonochromatic light from 560 to 800 nm, linearly polarized either parallel (0 degree) or perpendicular (90 degrees) to the fiber long axis. The signals from both dyes depend strongly on the incident polarization. At any wavelength and/or polarization condition, the total absorbance signal is a superposition of the same two signal components previously identified with unpolarized light (Heiny, J. A., and J. Vergara, 1982, J. Gen. Physiol., 80:203)--namely, a fast step signal from the voltage-clamped surface membrane and a signal reflecting the slower T-system potential changes. The 0 degree and 90 degrees spectra of both membranes have similar positive and negative absorbance peaks (720 and 670 nm, respectively, for dye NK2367; 740 and 700 nm for dye WW375); in addition, they have the same dichroic maxima (670 for NK2367; 700 for WW375). However, for the surface membrane, the 0 degrees spectra are everywhere more positive than the 90 degrees spectra, whereas the reverse is true for the T-system, which results in a dichroism of opposite sign for the two membranes. These spectral characteristics were analyzed using a general model for the potential-dependent response of an absorbing dye (Tasaki, I., and A. Warashina, 1976, Photochem. Photobiol., 24:191), which takes into account both the dye response and the membrane geometries. They are consistent with the proposal that the dye responds via a common mechanism in both membranes that consists of a dye reorientation and a change in the absorption maxima.     

The behavior of the fluorescence lifetime and polarization of oxonol potential-sensitive extrinsic probes in solution and in beef heart submitochondrial particles

Summary The fluorescence polarization and lifetime of the extrinsic potential-sensitive probes oxonols V and VI have been investigated both for the dyes free in aqueous and ethanol solutions and in the presence of beef heart submitochondrial particles under resting and energy-transducing conditions. The emission lifetime of the dyes appears to be inversely related to the solvent dielectric constant and increases as the solvent is changed from an aqueous medium to ethanol to the biological membrane. The fluorescence decay curve becomes biphasic in the presence of the membrane preparation and consists of a faster decaying component, the lifetime of which is the same as that of the probe in aqueous solution and of a slower decaying component. The longer lived component suffers an uncoupler-sensitive decrease in lifetime when ATP is added to the medium. The decrease in lifetime of the longer lived species is accompanied by large depolarizations of the dye fluorescence. These observations are consistent with a redistribution-type mechanism for the energy-dependent spectral changes involving the movement of probe from the aqueous phase to the membrane vesicles. The rotational relaxation time of oxonols V and VI is increased by over an order of magnitude when these dyes associate with the membrane. This observation is consistent with a previously developed model for the location of the dyes in the bilayer in which the side chains serve as anchors, preventing the rapid tumbling of the probe in the membrane.     

Calmodulin activation of the Ca2+ pump revealed by fluorescent chelator dyes in human red blood cell ghosts

Ca2+ transport in red blood cell ghosts was monitored with fura2 or quin2 incorporated as the free acid during resealing. This is the first report of active transport monitored by the fluorescent intensity of the chelator dyes fura2 (5-50 microM) or quin2 (250 microM) in hemoglobin-depleted ghosts. Since there are no intracellular compartments in ghosts and the intracellular concentrations of all assay chelator substances including calmodulin (CaM), the dyes, and ATP could be set, the intracellular concentrations of free and total Ca [( Cafree]i and [Catotal]i) could be calculated during the transport. Ghosts prepared with or without CaM rapidly extruded Ca2+ to a steady-state concentration of 60-100 nM. A 10(4)-fold gradient for Ca2+ was routinely produced in medium containing 1 mM Ca2+. During active Ca2+ extrusion, d[Cafree]i/dt was a second order function of [Cafree]i and was independent of the dye concentration, whereas d[Catotal]i/dt increased as a first order function of both the [Cafree]i and the concentration of the Ca:dye complex. CaM (5 microM) increased d[Catotal]i/dt by 400% at 1 microM [Cafree]i, while d[Cafree]i/dt increased by only 25%. From a series of experiments we conclude that chelated forms of Ca2+ serve as substrates for the pump under permissive control of the [Cafree]i, and this dual effect may explain cooperativity. Free Ca2+ is extruded, and probably also Ca2+ bound to CaM or other chelators, while CaM and the chelators are retained in the cell.     

Oxonol dyes as monitors of membrane potential. Their behavior in photosynthetic bacteria.

The reponses of oxonol dyes to single and multiple single turnovers of the photosynthetic apparatus of photosynthetic bacteria have been studied, and compared with the responses of the endogenous carotenoid pigments. The absorbance changes of the oxonols can be conveniently measured at 587 nm, because this is an isosbestic point in the 'light-minus-dark' difference spectrum of the chromatophores. The oxonols appear to respond to the light-induced 'energization' by shifting their absorption maxima. In the presence of K+, valinomycin abolished and nigericin enhanced such shifts, suggesting that the dyes, respond to the light-induced membrane potential. Since the dyes are anions at neutral pH values, they probably distribute across the membrane in accordance with the potential, which is positive inside the chromatophores. The accumulation of dye, which is indicated by a decrease in the carotenoid bandshift, poises the dye-membrane equilibrium in favor of increased dye binding and this might be the cause of the spectral shift. The dye response has an apparent second-order rate constant of approx. 2 . 10(6) M-1 . s-1 and so is always slower than the carotenoid bandshift. Thus the dyes cannot be used to monitor membrane potential on submillisecond timescales. Nevertheless, on a timescale of seconds the logarithm of the absorbance change at 587 nm is linear with respect to the membrane potential calibrated with the carotenoid bandshift. This suggests that under appropriate conditions the dyes can be used with confidence as indicators of membrane potential in energy-transducing membranes that do not possess intrinsic probes of potential.     

Oxonol dyes as monitors of membrane potential. Their behavior in photosynthetic bacteria

The responses of oxonol dyes to single and multiple single turnovers of the photosynthetic apparatus of photosynthetic bacteria have been studied, and compared with the responses of the endogenous carotenoid pigments. The absorbance changes of the oxonols can be conveniently measured at 587 nm, because this is an isosbestic point in the ‘light-minus-dark’ difference spectrum of the chromatophores.The oxonols appear to respond to the light-induced ‘energization’ by shifting their absorption maxima. In the presence of K⁺, valinomycin abolished and nigericin enhanced such shifts, suggesting that the dyes respond to the light-induced membrane potential. Since the dyes are anions at neutral pH values, they probably distribute across the membrane in accordance with the potential, which is positive inside the chromatophores. The accumulation of dye, which is indicated by a decrease in the carotenoid bandshift, poises the dye-membrane equilibrium in favor of increased dye binding and this might be the cause of the spectral shift.The dye response has an apparent second-order rate constant of approx. 2 · 10⁶ M^?1 · s^?1 and so is always slower than the carotenoid bandshift. Thus the dyes cannot be used to monitor membrane potential on submillisecond timescales. Nevertheless, on a timescale of seconds the logarithm of the absorbance change at 587 nm is linear with respect to the membrane potential calibrated with the carotenoid bandshift. This suggests that under appropriate conditions the dyes can be used with confidence as indicators of membrane potential in energy-transducing membranes that do not posses intrinsic probes of potential.     

Shortening of life-time of the pair [P680+Pheo-] contributes to general inhibitory effect of dinoseb on electron transfer in PS-II

It is shown that dinoseb, added to subchloroplast photosystem-II (PS-II) preparations from pea at a concentration higher than 5 microM, along with blocking the electron transfer on the acceptor side of PS-II, induces the following effects revealing its capability to have redox interaction with the components of PS-II reaction center (RC)-pheophytin (Pheo) and chlorophyll P680: (1) acceleration of the dark relaxation of absorbance (delta A) and chlorophyll fluorescence (delta F) changes related to photoreduction of Pheo as a result of the photoreaction [P680Pheo] [symbol: see text] [P680Pheo-] that leads to elimination of the delta A and delta F at a concentration of the inhibitor higher than 50 microM; (2) lowering of the maximum level of fluorescence (F) due to a decrease of delta F under the condition when the electron acceptor, QA, is reduced; (3) loss of the described effects of dinoseb and appearance of its capability to donate electron to RC of PS-II in the presence of dithionite which reduces dinoseb in the dark; (4) inhibition of delta A related to photooxidation of P680; (5) activation of delta A related to photooxidation P700 in photosystem-I (PS-I) preparations (a similar effect is observed upon the addition of 0.2 mM methylviologen). It is suggested that redox interaction with the pair [P680+Pheo-] leading to the shortening of its life-time contributes to the general effect of inhibition of electron transfer in PS-II by dinoseb.     

Spectral analyses of absorption changes associated with nerve excitation in dye-stained crab nerve

Spectral characteristics of absorption changes associated with nerve excitation were studied with crab nerves stained with a homologous series of dyes, merocyanine-rhodanines and rhodanine oxonols. In these classes of dyes, the absorption changes which followed approximately the same time course as that of the action potential (fast responses) depended in a similar fashion on the wavelength and polarization of the incident light. In order to interpret those commonly observed dependencies, a mode of reorientation of the absorption oscillators of the dye molecules in the membrane matrix during nerve excitation was proposed. In addition to the fast changes mentioned above, slow responses which developed during and after the action potential were commonly observed with oxonols. The spectra of the slow changes differed from those of the fast ones, indicating a distinct mechanism on the response production. A possible mechanism of the production of fast responses was also discussed based on the proposed mode of reorientation of the absorption oscillators.     

Comparative study of membrane potential-sensitive fluorescent probes and their use in ion channel screening assays

In this study, the authors compared and evaluated 4 membrane potential probes in the same cellular assay: the oxonol dye DiBAC(4)(3), the FLIPR membrane potential (FMP) dye (Molecular Devices), and 2 novel fluorescence resonance energy transfer (FRET) dye systems from PanVera [CC2-DMPE/DiSBAC(2)(3)] and Axiom [DiSBAC(1)(3)/DiSBAC(1)(5)]. The kinetic parameters of each membrane probe were investigated in RBL-2H3 cells expressing an endogenous inward rectifier potassium channel (IRK1). The FMP dye presented the highest signal over background ratio whereas the FRET dyes from PanVera gave the fastest response. The determination of IC(50) values for 8 different channel modulators indicated a good correlation between the 4 membrane probe systems. The compound-dye interaction was evaluated in the presence of compounds at 10 muM and clearly indicated no effect on the FMP or the PanVera donor dye, whereas some major interference with the oxonol probes was observed. Using a cell permeabilization assay in the presence of gramicidin, the authors concluded that the FRET dyes from PanVera and the FMP dye are unable to measure the gramicidin-induced cell membrane hyperpolarizations. The 4 dye systems were investigated under high-throughput screening (HTS) conditions, and their respective Z' parameter was determined. The characteristics of each dye system and its potential use in HTS assays is discussed.     

The mechanism of pantothenate transport by rat liver parenchymal cells in primary culture

The mechanism of pantothenate transport across the plasma membrane was investigated with initial velocity studies of [14C]pantothenate uptake and efflux in rat liver parenchymal cells maintained in primary culture. At 116 mM sodium, double-reciprocal plots of the initial velocity of uptake versus [pantothenate] were linear from 0.3 to 36.5 microM pantothenate and gave an apparent Km,pant of 11 +/- 2 microM. The rate of pantothenate uptake at 0 [sodium] was about 14% of the rate at 116 mM sodium, and the reciprocal of the apparent Km,pant was a linear function of [sodium]. Vmax obtained by extrapolation to infinite [pantothenate] was independent of [sodium]. Ouabain, gramicidin D, cyanide, azide, and 2,4-dinitrophenol inhibited uptake, but preloading cells with pantothenate did not. Pantothenate derivatives or carboxylic acids were only weak inhibitors of uptake. Efflux was measured in cells preloaded with [14C]pantothenate. The apparent Km for efflux was 85 +/- 29 microM, and the rate of efflux was unaffected by addition of pantothenate, sodium, ouabain, gramicidin D, or 2,4-dinitrophenol to the external medium. These features are consistent with a mechanism for pantothenate transport in which sodium and pantothenate are cotransported in a 1:1 ratio on a carrier highly specific for pantothenate; sodium decreases the apparent Km for pantothenate, and a sodium-carrier complex forms only on the intracellular side of the membrane.