A.P.R. Theuvenet

Sugar transport and potassium permeability in yeast plasma membrane vesicles

Plasma membrane vesicles were isolated from homogenised yeast cells by filtration, differential centrifugation and aggregation of the mitochondrial vesicles at pH 4. As judged by biochemical, cell electrophoretic and electron microscopic criteria a pure plasma membrane vesicle preparation was obtained. The surface charge density of the plasma membrane vesicles is similar to that of intact yeast cells with an isoelectric point below pH 3. The mitochondrial vesicles have a higher negative surface charge density in the alkaline pH range. Their isoelectric point is near pH 4.5, where aggregation is maximal. The yield of vesicles sealed to K+ was maximal at pH 4 and accounted for about one third of the total vesicle volume. The plasma membrane vesicles demonstrate osmotic behaviour, they shrink in NaCl solutions when loosing K+. As in intact yeast cells the entry and exit of sugars like glucose or galactose in plasma membrane vesicles is inhibited by UO22+. Counter transport in plasma membrane vesicles with glucose and mannose and iso-counter transport with glucose suggests that a mobile carrier for sugar transport exists in the plasma membrane. After galactose pathway induction in the yeast cells and subsequent preparation of plasma membrane vesicles the uptake of galactose into the vesicles increased by almost 100% over the control value without galactose induction. This increase is explained by the formation of a specific galactose carrier in the plasma membrane.

Besides affecting intracellular calcium signaling, 2-APB reversibly blocks gap junctional coupling in confluent monolayers, thereby allowing measurement of single-cell membrane currents in undissociated cells

2‐Aminoethoxydiphenyl borate (2‐APB) has been widely used as a blocker of the IP3 receptor and TRP channels, including store‐operated calcium channels. We now show in monolayers of normal rat kidney cells (NRK/49F) that 2‐APB completely and reversibly blocks gap junctional intercellular communication at concentrations similar to that required for inhibition of PGF2α‐induced increases in intracellular calcium. Gap junctional conductances between NRK cells were estimated with single‐electrode patch‐clamp measurements and were fully blocked by 2‐APB (50 µM), when applied extracellularly but not via the patch pipette. Half maximal inhibition (IC50) of electrical coupling in NRK cells was achieved at 5.7 µM. Similar results were obtained for human embryonic kidney epithelial cells (HEK293/tsA201) with an IC50 of 10.3 µM. Using 2‐APB as an electrical uncoupler of monolayer cells, we could thus measure inward rectifier potassium, L‐type calcium, and calcium‐dependent chloride membrane currents in confluent NRK monolayers, with properties similar to those in dissociated NRK cells in the absence of 2‐APB. The electrical uncoupling action described here is a new 2‐APB property that promises to provide a powerful pharmacological tool to study single‐cell properties in cultured confluent monolayers and intact tissues by electrical and chemical uncoupling of the cells without the need of prior dissociation.

Determination of gap junctional intercellular communication by capacitance measurements

Electrical coupling between cells is usually measured using the double patch-clamp technique with cell pairs. Here, a single patch-clamp technique that is not limited to cell pairs is described to determine electrical coupling between cells. Capacitance measurements in clusters of normal rat kidney (NRK) fibroblasts were used to study intercellular communication. In the whole-cell patch-clamp configuration capacitive transients were evoked by applying small voltage pulses. Total membrane capacitance was calculated from these capacitive transients after determination of access resistance, membrane conductance, and the decay constant of the transients, or alternatively by integrating the current transient. We found that in clusters of one to ten cells, membrane capacitance increased linearly with cell number, showing that the cells are electrically coupled. Membrane conductance of the cluster of cells also increased, as expected for cells that are well coupled. In subconfluent and confluent cultures, high membrane conductances together with large capacitive transients were observed, indicative of electrical coupling. Capacitance could only be determined qualitatively under these conditions, due to space clamp problems. In the presence of the gap junctional inhibitors halothane, heptanol or octanol, capacitance of all clusters of cells fell to single-cell levels, showing a complete uncoupling of the cells. The tumour promoter 12-O-tetradecanoylphor-bol-13-acetate (TPA) also uncoupled the cells completely, within 10 min. We conclude that capacitance measurements can provide a useful tool to study changes in intercellular communication in clusters of cells.

Kinetics of Ca2+ and Sr2+ uptake by yeast. Effects of pH, cations and phosphate.

The uptake of Ca2+ and Sr2+ by the yeast Saccharomyces cerevisiae is energy dependent, and shows a deviation from simple Michaelis-Menten kinetics. A model is discussed that takes into account the effect of the surface potential and the membrane potential on uptake kinetics. The rate of Ca2+ and Sr2+ uptake is influenced by the cell pH and by the medium pH. The inhibition of uptake at low concentration of Ca2+ and Sr2+ at low pH may be explained by a decrease of the surface potential. The inhibition of Ca2+ and Sr2+ uptake by monovalent cations is independent of the divalent cation concentration. The inhibition shows saturation kinetics, and the concentration of monovalent cation at which half-maximal inhibition is observed, is equal to the affinity constant of this ion for the monovalent cation transport system. The inhibition of divalent cation uptake by monovalent cations appears to be related to depolarization of the cell membrane. Phosphate exerts a dual effect on uptake of divalent cations: and initial inhibition and a secondary stimulation. The inhibition shows saturation kinetics, and the inhibition constant is equal to the affinity constant of phosphate for its transport mechanism. The secondary stimulation can only partly be explained by a decrease of the cell pH, suggesting interaction of intracellular phosphate, or a phosphorylated compound, with the translocation mechanism.

Kinetics of sulfate uptake by yeast

Uptake of sulfate by yeast requires the presence of a metabolic substrate and is dependent on the time during which the cells have been metabolizing in the absence of sulfate. At low concentrations of sulfate, uptake can be described by simple saturation kinetics. Uptake of sulfate is accompanied by a net proton influx of 3 H+ and an efflux of 1 K+ for each sulfate ion taken up. Divalent cations stimulate sulfate uptake at low concentrations of sulfate; the maximal rate of uptake is not significantly affected but Km is lowered. Stimulation by divalent cations shows an optimum at a cation concentration of about 4 mM. Monovalent cations are less effective, trivalent cations are more effective in stimulating sulfate uptake. The results are qualitatively in accordance with the notion, that the effect of cations is due to an effect via the surface potential.

Kinetics of ion translocation across charged membranes mediated by a two-site transport mechanism: Effects of polyvalent cations upon rubidium uptake into yeast cells

(1) The effect of surface charge upon the kinetics of monovalent cation translocation via a two-site mechanism is investigated theroretically. (2) According to the model dealt with, typical relations are expected for the dependence of the kinetic parameters of the translocation process upon the concentration of a polyvalent cation, differing essentially from those derived for the case in which the membrane carries no excess charge. (3) Even when a polyvalent cation does not compete with the substrate cation for binding to the translocation sites, apparently competitive inhibition may occur when the membrane is negatively charged. (4) The model is tested experimentally by studying the effects of the polyvalent cations Mg2+, Sr2+, Ca2+, Ba2+ and Al3+ upon Rb+ uptake into yeast cells at pH 4.5 A good applicability is found. (5) Equimolar concentrations of polyvalent cations reduce the rate of the Rb+ uptake into yeast cells in the order Mg2+ less than Sr2+ less than Ca2+ less than Ba2+ less than Al3+. (6) The conclusion is reached that the reduction in the rate of Rb+ uptake caused by the polyvalent cations applied results mainly from screening of the negative fixed charges on the membrane surface and binding to these negative sites rather than competition with Rb+ for the transport sites. (7) The results of our investigation indicate the affinity of the alkaline-earth cations for the negative fixed charges on the surface to the yeast cell membrane increases in the orther Mg2+ less than Sr2 less than Ca2+ less than Ba2+. (8) Probably mainly phosphoryl groups determine the net charge on the membrane of the yeast cell at a medium pH of 4.5.

Synchronized calcium spiking resulting from spontaneous calcium action potentials in monolayers of NRK fibroblasts

The correlation between the intracellular Ca2+ concentration ([Ca2+]i) and membrane potential in monolayers of density-arrested normal rat kidney (NRK) fibroblasts was investigated. Using the fluorescent probe Fura-2, spontaneous repetitive spike-like increases in [Ca2+]i (Ca2+ spikes) were observed that were synchronised throughout the entire monolayer. Ca2+ spikes disappeared in Ca(2+)-free solutions and could be blocked by the L-type Ca2+ channel antagonist felodipine. Simultaneous measurements of [Ca2+]i and membrane potential showed that these Ca2+ spikes were paralleled by depolarisations of the plasma membrane. Using patch clamp measurements, action potential-like depolarisations consisting of a fast spike depolarisation followed by a plateau phase were seen with similar kinetics as the Ca2+ spikes. The action potentials could be blocked by L-type Ca2+ channel blockers and were dependent on extracellular Ca2+. The plateau phase was predominantly determined by a Cl- conductance and was dependent on intracellular Ca2+. The presence of voltage-dependent L-type Ca2+ channels in NRK cells was confirmed by patch clamp measurements in single cells. It is concluded that monolayers of density-arrested NRK fibroblasts exhibit spontaneous Ca2+ action potentials leading to synchronised Ca2+ spiking. This excitability of monolayers of fibroblasts may represent a novel Ca2+ signaling pathway in electrically coupled fibroblasts, cells that were hitherto considered to be inexcitable.

Hysteresis and bistability in a realistic cell model for calcium oscillations and action potential firing.

Many cells reveal oscillatory behavior. Some cells reveal action-potential firing resulting from Hodgkin-Huxley (HH) type dynamics of ion channels in the cell membrane. Another type of oscillation relates to periodic inositol triphospate (IP3)-mediated calcium transients in the cytosol. In this study we present a bifurcation analysis of a cell with an excitable membrane and an IP3-mediated intracellular calcium oscillator. With IP3 concentration as a control parameter the model reveals a complex, rich spectrum of both stable and unstable solutions with hysteresis corresponding to experimental data. Our results reveal the emergence of complex behavior due to interactions between subcomponents with a relatively simple dynamical behavior.

Cadmium ion is a non-competitive inhibitor of red cell Ca2+-ATPase activity

In the presence as well as in the absence of calmodulin, Cd2+ inhibits the human erythrocyte plasma membrane Ca(2+)-ATPase activity non-competitively with Ki = 2 nM, whereas ATP-dependent Ca(2+)-transport across the red cell membrane was found to be inhibited competitively by Cd2+ (Verbost, P.M., Flik, G., Pang, P.K.T., Lock, R.A.C. and Wendelaar Bonga, S.E. (1989) J. Biol. Chem. 264, 5613-5615). In this study it will be argued that Cd2+ also inhibits Ca(2+)-transport non-competitively, and that the discrepancy with previous conclusions most probably relies on use of an incorrect computer program that calculates the free concentrations of Ca2+ and Cd2+ at the experimental conditions applied for measurement of Ca2+ uptake.

Effect of surface potential on Rb+ uptake in yeast: The effect of pH

Abstract The apparent K m of Rb + uptake and the zeta potential of yeast cells are appreciably affected by changes in the pH, variation of the concentration of the buffer cation Tris + and addition of Ca 2+ to the suspending medium. Irrespective of the way in which the zeta potential is affected, a direct relationship between the apparent K m of the Rb + uptake and the zeta potential is observed. A reduction of 8 mV in the zeta potential is accompanied by a 20-fold increase in the apparent K m , which illustrates that electrostatic effects in ion uptake cannot be ignored. Measured zeta potentials are, to a good approximation, linearly related to surface potentials evaluated from a kinetic analysis of the Rb + uptake. This shows the practical use of the zeta potential as a measure of the surface potential in studies of electrostatic effects in ion uptake by yeast. It is concluded that Tris + and the aikaline earth cations inhibit the Rb + uptake in yeast exclusively via a reduction in the surface potential. Protons, in addition, exert a competitive inhibition.