Friedrich-Wilhelm Bentrup

Relationship between cell turgor pressure, electrical membrane potential, and chloride efflux inAcetabularia mediterranea

SummaryTurgor pressureP, electrical membrane potentialVm and release of36Cl− have been measured on individual cells of the marine green algaAcetabularia mediterranea. In contrast to other marine and pond water algal cells a continuous turgor pressure increase is observed in many cells ofA. mediterranea (0.8 to 5×10−4 bar sec−1), even though the osmolarity of the external seawater is kept constant. Above a critical pressure value of 2.26±0.22 bar (n=25,T=18 and 23 °C), the turgor pressure exhibits continuous decreases and increases in a range of 0.22±0.13 bar (n=23). At lower temperatures (6 to 10 °C) the threshold for pressure regulation is shifted towards higher values. Pressure regulations in the critical range are accompanied by bursts of36Cl-efflux and in most cases by action potentials, but not every action potential is associated with a decrease in turgor pressure. The frequency of spontaneous action potentials increases with rising cell turgor. The amount of36Cl-released, i.e. about 4% of the cellular Cl− content (H. Mummert, Ph.D. Thesis, University of Tübingen, Germany, 1979), compares well with the observed reduction in turgor pressure after the burst. The kinetics of turgor reduction, however, are not compatible with a release of chloride by vesicular transport as suggested by H. Mummert and D. Gradmann (In: Plant Membrane Transport: Current Conceptual Issues. Elsevier/ North-Holland, Amsterdam, 1980). It is concluded that the steady turgor increase is generated by a net influx consisting largely of KCl due to the electrogenic chloride import pump and the strong inward driving force for K+ whereas the subsequent occurring regulations of turgor pressure can be understood on the basis of a passive chloride channel in the plasmalemma which is controlled by turgor.

Substrate specifity of the hexose carrier in the plasmalemma of Chenopodium suspension cells probed by transmembrane exchange diffusion

Substrate specifity of the proton-driven hexose cotransport carrier in the plasmalemma of photoautotrophic suspension cells of Chenopodium rubrum L. has been studies through the short-term perturbation of 14C-labelled efflux of 3-O-methyl-d-glucose. Efflux, occurring exclusively via carrier-mediated exchange diffusion, is trans-stimulated by the substrate and trans-inhibited by the glucose-transport inhibitors phlorizin (K1/2=7.9 mM) and its aglucon phloretin (K1/2=84 μM); with both inhibitors, 3-O-methyl-d-glucose efflux may be blocked completely. Trans-stimulation of efflux (up to fourfold) by a variety of the d-enantiomers of neutral hexoses, including glucose (K1/2=48 μM), 3-O-methyl-d-glucose (K1/2=139 μM), and fructose (K1/2=730 μM), but not by, for instance, d-allose, and l-sorbose, shows that carrier-substrate interaction critically involves the axial position at C-1 and C-3, respectively. We suggest that substrate binding by the Chenopodium hexose carrier involves both hydrophobic interaction with the pyran-ring and hydrogen-ion bonding at C-1 and C-3 of the d-glucose conformation.

Potassium ion channels in the plasmalemma

The potassium ion is an indispensible cytosolic component of living cells and a key osmolyte of plant cells, crossing the plasmalemma to drive physiological processes like cell growth and motor cell activity. K(+) transport across the plasmalemma may be passive through channels, driven by the electrochemical gradient, K(+) equilibrium potential (E(K) ) - membrane potential (V(m) ), or secondary active by coupling through a carrier to the inward driving force of H(+) or Na(+) . Known K(+) channels are permeable to monovalent cations, a permeability order being K(+) > Rb(+) > NH(4) (+) > Na(+) ≥ Li(+) > Cs(+) . The macroscopic K(+) currents across a cell or protoplast surface commonly show rectification, i.e. a V(m) -dependent conductance which in turn, may be controlled by the cytosolic activity of Ca(2+) , of K(+) , of H(+) , or by the K(+) driving force. Analysis by the patch clamp technique reveals that plant K(+) channels are similar to animal channels in their single channel conductance (4 to 100 pS), but different in that a given channel population slowly activates and may not inactivate at all. Single-channel kinetics reveal a broad range of open times (ms to s) and closed times (up to 100 s). Further progress in elucidating plant K(+) channels will critically depend on molecular cloning, and the availability of channel-specific (phyto)toxins.

Kinetics and Specificity of ATP-dependent Proton Translocation Measured with Acridine Orange in Microsomal Fractions from Green Suspension Cells of Chenopodium rubrum L.

From photoautotrophic cell suspension cultures of Chenopodium rubrum L. microsomal fractions were prepared by differential and isopycnic sucrose gradient centrifugation. ATP-generated proton-accumulation was studied on a microsomal fraction (MF) of proton-translocating vesicles with a density of 1.11 to 1.12 gem(-3). This activity did not overlap with mitochondrial or chloroplast markers. MF is presumably of tonoplast origin. The assay was based upon pH-dependent acridine orange (AO) absorption shifts at 490 nm, Δ(490), due to AO-accumulation and polymerization within the vesicles, and was calibrated using pK and K(D) for AO polymers given by Zanker (Z. physikal. Chemie 199, 225, 1952). Addition of 1 mM ATP to MF yields a transmembrane ΔpH of about 2 units within 10 min (kinetics with a single time constant, τ=400s) and collapses after addition of the protonophor CCCP (τ=22s). The proton pumping activity is insensitive to vanadate and highly specific for ATP compared with GTP, UTP, CTP, and ITP, and half-saturates at 0.38 to 0.46 mM ATP, indicated by both initial rate (1 min) and steady state value of Δ(490) (10 min). Proton pumping into and proton leakage from the vesicles was cast into a feedback-scheme showing that the pump is working at a constant rate, that is, independently of the size of the created ΔpH.

Calmodulin regulates the Ca2+-dependent slow-vacuolar ion channel in the tonoplast of Chenopodium rubrum suspension cells

The patch-clamp technique was applied to vacuoles isolated from a photoautotrophic suspension cell culture of Chenopodium rubrum L. and vacuolar clamp currents, which are predominantly carried by the previously identified Ca2+-dependent slow vacuolar (SV) ion channels, were recorded. These currents, which were activated by 1-s voltage pulses of -100 mV (vacuolar interior negative) in the presence of 100 μM Ca2+ (cytosolic side), could be blocked completely and reversibly by the calmodulin antagonist W-7 [N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide] and its chlorine-deficient analogue W-5; half-maximum inhibition was found at approx. 6 μM for W-7 and 70 μM for W-5. Inhibition was reversed by addition of 1 μg · ml−1 calmodulin purified from Chenopodium cell suspensions; reversal by bovine brain calmodulin was scarcely appreciable. We conclude that cytosolic calmodulin mediates the Ca2+ dependence of the SV-channel in the Chenopodium tonoplast.

Phlorizin inhibits hexose transport across the plasmalemma of Riccia fluitans.

The effect of phlorizin has been tested on hexose transport and hexose-induced changes of electrical potential (ψm) and conductance (gm) across the plasmalemma of rhizoid and thallus cells of the aquatic liverwort Riccia fluitans. The decrease of ψm (depolarization) and gm induced by 1 mM 3-oxymethyl-D-glucose (3-OMG) is substantially inhibited by simultaneous addition of 2 mM phlorizin, whereas no significant response was observed when phlorizin was added alone or several minutes after the sugar. Current-voltage data show that the 3-OMG-generated electrical inward current of 0.016 A m-2 drops to 0.010 A m-2 when phlorizin is present. Uptake as well as efflux of [14C]-3-OMG is strongly and reversibly inhibited by phlorizin between 0.2 and 5 mM. The results are consistent with our hypothesis that the hexose carrier has one binding site with competitive inhibition of glucose uptake by phlorizin (ki=0.08 mM). The electrical data indicate that phlorizin affects an ψm step of the carrier transport cycle.

Pharmacology of the SV channel in the vacuolar membrane of Chenopodium rubrum suspension cells.

Single channel performance and deactivation currents have been analyzed in the presence of cation channel blockers to reveal pharmacological properties of the slow-activating (SV) cation-selective ion channel in the vacuolar membrane (tonoplast) isolated from suspension cells of Chenopodium rubrum L. At a holding potential of −100 mV, the SV channel showed half-maximal inhibition with 20mm tetraethylammonium (TEA), 7 μm 9amino-acridine, 6 μm (+)-tubocurarine, 300nm quinacrine, and 35 μm quinine, respectively. The SV channel is also blocked by charybdotoxin (20nm at −80 mV) but not by apamine. 9-Amino-acridine, (+)-tubocurarine and quinacrine act in a voltage-dependent fashion, binding to the open channel and to different sites along the transmembrane voltage profile according to Woodhull (J. Gen. Physiol.61:687–708, 1973). No binding site could be specified for charybdotoxin, which binds to the closed channel, and for quinine. Except for quinine, all tested blockers were effective only if added to the cytoplasmic side of the tonoplast. A structural relationship between the SV channel and Maxi-K channels in animal systems is inferred.

Voltage- and Ca2+-dependence of the K+ channel in the vacuolar membrane of Chenopodium rubrum L. suspension cells

Voltage- and Ca(2+)-dependence of the slow-activating SV-K+ channel in the vacuolar membrane of Chenopodium rubrum suspension cells has been analyzed using the patch clamp technique in the vacuole-attached, outside-out and whole-vacuolar configuration. Patch-pipette perfusion was applied to measure Ca2+ dependence of single channels in the attached-configuration. Using the PCLAMP-software (Axon Instruments), an algorithm was developed to extract reliable individual channel data from multi-channel activity records, including open probability, mean open and closed times, as well as time constants for open and closed distributions. The channel conductance of the major open state was about 83 pS (seal resistance > 8 G omega) at 30 mV (transmembrane voltage Vm, vacuole negative), and symmetrical 100 mM KCl. the channel exhibited a strong voltage- and a weak Ca(2+)-activation: increasing Vm from 40 to 100 mV is equivalent to a Ca2+ concentration change from 10(-7) to 10(-4) M. Mean open probabilities at Vm = 30 mV were 0.03 with 1 microM and 0.09 with 100 microM Ca2+. Mean open times were approx. 7 ms, and almost independent of both, voltage and Ca2+. Mean closed times, however, varied in a strongly voltage- and Ca(2+)-dependent manner, e.g., at Vm = 30 mV dropped from 205 to 67 ms, if Ca2+ was raised from 10(-6) to 10(-4) M. Open and closed distributions of events within bursts could be fitted by the sum of two exponentials with time constants between 0.3 and 11 ms.

AC fields of low frequency and amplitude stimulate pollen tube growth possibly via stimulation of the plasma membrane proton pump

Abstract Growing pollen tubes of Lilium longiflorum Thunb. were exposed to weak AC fields (1.75–100 mV cm−1) of low frequency (1–100 Hz). A stimulation of tube growth was observed in a narrow range of AC fields (1.75–30 mV cm−1 and 1–20 Hz) with a maximum effect (1.42-fold stimulation) at 20 mV cm−1 and 10 Hz. Pollen tubes did not change their growth direction during field application. The stimulation was independent from the orientation of the tubes to the field, but depended on the initial growth rate. Slowly growing pollen tubes (1–3 μm min−1) were more stimulated than fast growing tubes (> 6 μm min−1). In search for a possible target interacting with the AC field we focused on the P-type H+ ATPase of the plasma membrane. H+ transport activity of a microsomal fraction obtained from pollen tubes was also stimulated by AC fields showing a maximum stimulation at similar AC fields as tube growth (10 mV cm−1, 10 Hz). The stimulation of H+ transport was still observed in the presence of azide and bafilomycin inhibiting the mitochondrial, F-type H+ ATPase and the vacuolar, V-type H+ ATPase, respectively. No stimulation was observed in the presence of the P-type H+ ATPase inhibitor vanadate. We, therefore, suggest that an increased activity of the plasma membrane H+ ATPase causes the increase in tube growth when pollen tubes were exposed to AC fields. These results underline the importance of the H+ ATPase in pollen tube growth; regulation of its activity may also regulate tube elongation in vivo.

Charybdotoxin blocks cation-channels in the vacuolar membrane of suspension cells of Chenopodium rubrum L.

Using the patch-clamp technique, we studied the action of charybdotoxin which blocks Ca(2+)-activated large-conductance K+ channels in animal tissue on the slow-activating (SV), Ca(2+)-activated cation channel in the vacuolar membrane of suspension-cells of Chenopodium rubrum L. The toxin reversibly reduced the vacuolar current with EC50 approximately 20 nM suggesting structural similarities between ion channels in animal and plant membranes.