Vacuolar calcium channels.

The central vacuole is the largest Ca2+ store in a mature plant cell. Ca2+ release from this store contributes to Ca2+-mediated intracellular signalling in a variety of physiological responses. However, the routes for vacuolar Ca2+ release are not well characterized. To date, at least two voltage-dependent and two ligand-gated Ca2+-permeable channels have been reported in plant vacuoles. However, the so-called VVCa (vacuolar voltage-gated Ca2+) channel most probably is not a separate channel but is identical to another voltage-dependent channel-the so-called SV (slow vacuolar) channel. Studies in the last few years have added a new dimension to our knowledge of SV channel-mediated ion transport and the mechanisms of its regulation by multiple natural factors. Recently, the SV channel was identified as the product of the TPC1 gene in Arabidopsis. In contrast, the TPC1 channel from other species was thought to be localized in the plasma membrane. A re-evaluation of this work under the assumption that the TPC1 channel is generally a vacuolar channel provides interesting insights into the physiological function of the TPC1/SV channel. Considerably less is known about vacuolar Ca2+ channels that are supposed to be activated by inositol 1,4,5-trisphosphate or cADP ribose. The major problems are controversial reports about functional characteristics, and a remarkable lack of homologues of animal ligand-gated Ca2+ channels in higher plants. To help understand Ca2+-mediated intracellular signalling in plant cells, a critical update of existing experimental evidence for vacuolar Ca2+ channels is presented.

Cooperative interaction of high-potential hemes in the cytochrome subunit of the photosynthetic reaction center of bacterium Ectothiorhodospira shaposhnikovii.

Cooperative interaction of the high-potential hemes (C(h)) in the cytochrome subunit of the photosynthesizing bacterium Ectothiorhodospira shaposhnikovii was studied by comparing redox titration curves of the hemes under the conditions of pulse photoactivation inducing single turnover of electron-transport chain and steady-state photoactivation, as well as by analysis of the kinetics of laser-induced oxidation of cytochromes by reaction center (RC). A mathematical model of the processes of electron transfer in cytochrome-containing RC was considered. Theoretical analysis revealed that the reduction of one heme C(h) facilitated the reduction of the other heme, which was equivalent to a 60 mV positive shift of the midpoint potential. In addition, reduction of the second heme C(h) caused a three- to four-fold acceleration of the electron transfer from the cytochrome subunit to RC.

Regulation of the slow vacuolar channel by luminal potassium: role of surface charge.

Voltage-dependent activation of slow vacuolar (SV) channels has been studied on isolated patches from red beet (Beta vulgaris L.) vacuoles. Isoosmotic variation of vacuolar K(+) from 10 to 400 mM in Ca(2+)-free solutions at the vacuolar side shifted the SV channel activation threshold to more positive voltages. The effect of K(+) could be mimicked by additions of choline or N-methyl D-glucamine and could be explained by unspecific screening of the negative surface charge. Fitting the dependence of voltage shift on K(+) concentration to the Gouy-Chapman model yields a surface charge density of 0.36 +/- 0.05 e(-)/nm(2). Negative surface potential also tended to increase the local concentration of permeable ions (K(+)), resulting in anomalously high single-channel conductance, approximately 200 pS in 10 mM KCl. An increase of ionic strength due to addition of impermeable cations greatly reduced the unitary conductance. Large positive shift of the SV channel voltage dependence, caused by physiological (0.5 mM) free vacuolar Ca(2+), was partly ameliorated by increasing luminal K(+). We interpreted these results as follows: K(+)induced a reduction of surface potential, hence i) causing a positive shift of the voltage dependence and ii) a dilution of Ca(2+) in the membrane vicinity, thus reducing the inhibitory effect of vacuolar Ca(2+) and causing a negative shift of the SV channel voltage dependence, with a sum of the two shifts being negative.

Fast-activating channel controls cation fluxes across the native chloroplast envelope.

A prerequisite for photosynthetic CO(2) fixation is the maintenance of alkaline pH in the stroma. This is achieved by H(+) pumping from the stroma to the cytosol, electrically balanced by an influx of cations through some unidentified non-selective envelope channels. In this study, the patch-clamp technique was applied to isolated Pisum sativum L. (pea) chloroplasts, and a fast-activating chloroplast cation (FACC) channel was discovered in the native envelope. This channel opens within a few milliseconds upon voltage steps to large positive or negative potentials. Remarkably, the single-channel conductance increased fivefold, from approximately 40 pS to approximately 200 pS (symmetric 250 mM KCl), upon a potential change from zero to +/- 200 mV. The FACC channel conducts all physiologically essential inorganic cations (K(+), Na(+), Ca(2+), Mg(2+)) with little preference. An increase of stromal pH from 7.3 to 8.0, mimicking dark-light transition, caused about a 2-fold decrease of the FACC channel activity within a physiologically relevant potential range. The FACC channel was completely and irreversibly blocked by Gd(3+). Based on the estimated transport capacity of the whole chloroplast population of FACC channels together with the envelope H(+)-ATPases, these channels can mediate electroneutral K(+)/H(+) exchange across the envelope, enabling stroma alkalinization, thereby allowing an optimal photosynthetic performance.

Conduction of monovalent and divalent cations in the slow vacuolar channel.

The conduction properties of individual physiologically important cations Na+, K+, Mg2+, and Ca2+ were determined in the slowly activating (SV) channel of sugar beet vacuoles. Current-voltage relationships of the open channel were measured on excised tonoplast patches in a continuous manner by applying a +/-140 mV ramp-wave protocol. Applying KCl gradients of either direction across the patch we have determined that the relative Cl- to K+ permeability was < or =1%. Symmetrical increase of the concentration of tested cation caused an increase of the single channel conductance followed by saturation. Fitting of binding isotherms at zero voltage to the Michaelis-Menten equation resulted in values of maximal conductance of 300, 385, 18, and 13 pS, and of apparent dissociation constants of 64, 103, 0.04, and 0.08 mm for Na+, K+, Mg2+, and Ca2+, respectively. Deviations from the single-ion occupancy mechanism are documented, and alternative models of permeation are discussed. The magnitude of currents carried by divalent cations at low concentrations can be explained by an unrealistically wide (approximately 140 A) radius of the pore entrance. We propose instead a fixed negative charge in the pore vestibules, which concentrates the cations in their proximity. The conduction properties of the SV channel are compared with reported characteristics of voltage-dependent Ca2+-permeable channels, and consequences for a possible reduction of postulated multiplicity of Ca2+ pathways across the tonoplast are drawn.

Asymmetric block of the plant vacuolar Ca(2+)-permeable channel by organic cations.

In this work we have analysed the voltage-dependent block of the slow activating channel from red beet vacuoles by Tris, quaternary ammonium ions and the natural polyamines putrescine, spermidine and spermine. All these organic cations when applied from the cytosolic side blocked the channel by binding apparently deep (zdelta values in the range of 0.65-1.35) within the pore. Tetraethylammonium ion did not pass the selectivity filter, whereas the cations with a smaller cross-section and Tris could pass across the entire pore, as evidenced by a relief of block at high positive voltages. Voltage dependence of the establishment of block from cytosolic side and of its relief was anomalously strong in the sense that the total charge moved across the pore for all blockers tested, with a notable exception of spermine, was in excess of their actual valence. This behaviour is consistent with the existence of multiple binding sites within a long pore, their simultaneous occupancy and interaction between different ions. In contrast, binding of blockers from the vacuolar (lumenal) side appears to follow a single-ion handling rule, with a common binding site for all amines located at approximately 30% of the electrical distance from the lumenal side.

Cooperative block of the plant endomembrane ion channel by ruthenium red.

Effects of ruthenium red (RR) on the slow Ca(2+)-activated Ca(2+)-permeable vacuolar channel have been studied by patch-clamp technique. Applied to the cytosolic side of isolated membrane patches, RR at concentrations of 0.1-5 microM produced two distinct effects on single channel kinetics, long lasting closures and a flickering block of the open state. The first effect was largely irreversible, whereas the second one could be washed out. The extent of flickering block steeply increased (zdelta = approximately 1.35) with the increase of cytosol-positive voltage, dragging RR into the channel pore. At least two RR ions are involved in the block according to Hill coefficient n = approximately 1.30 for the dose response curves. The on-rate rate of the drug binding linearly depended on the RR concentration, implying that one RR ion already plugged the pore. The blocked state was further stabilized by binding of the second RR. This stabilization was in excess of that predicted by independent binding as the dependence of unblocking rate on RR concentration revealed. A cooperative model was therefore employed to describe the kinetic behavior of RR binding. At zero voltage the half-blocking RR concentration of 36 microM and the bimolecular on-rate constant of 1.8 x 10(8) M(-1) s(-1) were estimated.

Inhibition of vacuolar ion channels by polyamines.

In this work, direct effects of cytosolic polyamines on the two principle vacuolar ion channels were studied by means of patch-clamp technique. Fast and slow activating vacuolar channels were analyzed on membrane patches isolated from vacuoles of the red beet taproot. The potency of the fast and of the slow vacuolar channel blockage by polyamines decreased with a decrease of the polycation charge, spermine4+ > spermidine3+ > putrescine2+. In contrast to the inhibition of the fast vacuolar channel, the blockage of the slow vacuolar channel by polyamines displayed a pronounced voltage-dependence. Hence, in the presence of high concentration of polyamines the slow vacuolar channel was converted into a strong inward rectifier as evidenced by its unitary current-voltage characteristic. The blockage of the slow vacuolar channel by polyamines was relieved at a large depolarization, in line with the permeation of polyamines through this channel. The voltage-dependence of blockage was analyzed in terms of the conventional model, assuming a single binding site for polyamines within the channel pore. Taking advantage of a simple linear structure of naturally occurring polyamines, conclusions on a possible architecture of the slow vacuolar channel pore were drawn. The role of common polyamines in regulation of vacuolar ion transport was discussed.

Ion channel permeable for divalent and monovalent cations in native spinach thylakoid membranes.

A cation-selective channel was characterized in isolated patches from osmotically swollen thylakoids of spinach (Spinacea oleracea). This channel was permeable for K+ as well as for Mg2+ and Ca2+ but not for Cl-. When K+ was the main permeant ion (symmetrical 105 mM KCl) the conductance of the channel was about 60 pS. The single channel conductance for different cations followed a sequence K+ > Mg2+ >/= Ca2+. The permeabilities determined by reversal potential measurements were comparable for K+, Ca2+, and Mg2+. The cation channel displayed bursting behavior. The total open probability of the channel increased at more positive membrane potentials. Kinetic analysis demonstrated that voltage dependence of the total open probability was determined by the probability of bursts formation while the probability to find the channel in open state within a burst of activity was hardly voltage-dependent. The cation permeability of intact spinach thylakoids can be explained on the single channel level by the data presented here.

Patch clamp study of the voltage-dependent anion channel in the thylakoid membrane.

Measurements of single channel currents were performed on isolated membrane patches from osmotically swollen thylakoids of the Charophyte alga Nitellopsis obtusa. A channel with a high selectivity for anions over cations and a conductance of 100 to 110 pS (114 mm Cl-) was revealed. The channel has a bell-shaped voltage-dependence of the open probability, with a maximum at about 0 mV. This dependence was explained by two gating processes, one causing channel closure at positive and one at negative potentials. The steepness of the voltage-dependence corresponded to approximately 2 elementary charges to be transferred across the entire membrane in each of the two gating processes. The analysis of the anion channel kinetics in the millisecond time domain revealed an e-fold increase of mean open and decrease of mean closed times when the membrane voltage was made more positive by 20 and 36 mV, respectively. Concert transitions of two identical anion channels between open and long inactivated states were observed, while the millisecond closed-open transitions of the two channels within a burst of activity were kinetically independent.

Patch-clamp study of vascular plant chloroplasts: ion channels and photocurrents.

This article presents direct measurements of large-conductance cation channel (G = 730 pS in 100/50 mM KCl; PK+/PCl- = 2.8) and light-induced current (photocurrent) in chloroplasts from C4 plants, Amaranthus hybridus and Zea mays, using the conventional patch-clamp technique. It was shown that preillumination of chloroplast gave rise to a fast decaying transient (tau approximately equal to 6 ms) in the light-induced current for the second and following light pulses. This transient increase of photocurrent was interpreted as a consequence of photoreducing of the redox pool between photosystems.

Depolarization-Activated K+ Channel in Chara Droplets.

A novel potassium channel was characterized in the droplet membrane of Chara gymnophylla. This channel has a conductance of about 90 pS (in symmetrical 0.15 M KCl), which is lower compared to the 170-pS K+ channel predominant in this preparation. In contrast to the large conductance K+ channel, the novel channel opened with a delay at depolarization and closed at hyperpolarization and did not require cytosolic Ca2+ for its opening. It also showed comparatively weak selectivity for K+ over other monovalent cations, although its cation to anion selectivity was high. Externally or internally applied Cs+ blocked the channel in a voltage-dependent manner, similarly to the 170-pS channel. The sensitivity of the 90-pS channel to external tetraethylammonium chloride (half-blocking concentration approximately 1.5 mM) was 20-fold higher compared to the large conductance channel. With respect to its voltage-gating kinetics, the 90-pS channel was identified as a "slow delayed rectifier."

Effects of D2O on permeation and gating in the Ca(2+)-activated potassium channel from Chara.

We studied the effects of H2O/D2O substitution on the permeation and gating of the large conductance Ca(2+)-activated K+ channels in Chara gymnophylla droplet membrane using the patch-clamp technique. The selectivity sequence of the channel was: K+ > Rb+ >> Li+, Na+, Cs+ and Cl-. The conductance of this channel in symmetric 100 mM KCl was found to be 130 pS. The single channel conductance was decreased by 15% in D2O as compared to H2O. The blockade of channel conductance by cytosolic Ca2+ weakened in D2O as a result of a decrease in zero voltage Ca2+ binding affinity by a factor of 1.4. Voltage-dependent channel gating was affected by D2O primarily due to the change in Ca2+ binding to the channel during the activation step. The Hill coefficient for Ca2+ binding was 3 in D2O and around 1 in H2O. The values of the Ca2+ binding constant in the open channel conformation were 0.6 and 6 microM in H2O and D2O, respectively, while the binding in the closed conformation was much less affected by D2O. The H2O/D2O substitution did not produce a significant change in the slope of channel voltage dependence but caused a shift as large as 60 mV with 1 mM internal Ca2+.

One of the chloroplast envelope ion channels is probably related to the mitochondrial VDAC.

The voltage dependence of large conductance channels in the intact chloroplast envelope of Nitellopsis obtusa was examined using the patch-clamp technique. The channel switched to the lower conducting substates with amplitudes of around 45 and 20% of that in the open state when potentials larger than 30 mV were applied. The steepness of the voltage dependence approximately corresponds to 4 elementary charges being transferred across the entire voltage drop to close the channel both at positive and negative potentials. The transition to the closed substates could also be induced by König's polyanion, a well known modulator of the mitochondrial outer membrane channel.

Single channel recording in the chloroplast envelope.

The patch-clamp technique was applied to study ion channels in the intact chloroplast envelope. Three channel types were characterized: two cation-selective, with a conductance (in 100 mM KCl) of 517 and 1016 pS, respectively, and one anion-selective with a conductance of 159 pS. All three channels showed voltage-dependent closures at both positive and negative membrane potentials.

Probing of pore in the Chara gymnophylla K+ channel by blocking cations and by streaming potential measurements.

The current-voltage (I-V) relationship of single K+ channels present in the Chara gymnophylla droplet membrane was studied. The channel presumably contains large mouths at both pore ends which are sufficiently wide to accommodate TEA+ as evidenced by internal and external TEA+ blockade. The voltage dependence of blockade by external Cs+ and Na+ reveals the multi-ion occupancy of the channel. The value of streaming potential (4.0 mV/osmol) measured in the Chara K+ channel indicates that the channel contains up to nine water molecules in the narrow region. It is concluded that the length of this region is around 28 A.

[Effect of dehydration on electron transport from membrane-bound cytochromes c to bacteriochlorophyll of the photosynthetic reaction center in chromatophores of purple thiobacteria]. / Vliianie degidratatsii na perenos élektrona ot sviazannykh s membranoi tsitokhromov c na bakteriokhlorofill fotosinteticheskogo reaktsionnogo tsentra v khromatoforakh purpurnykh serobakterii.

A method is proposed for spectroscopic probing photo-induced reversible oxidation-reduction changes of high-potential cytochrome in chromatophore films of various humidity. On these preparations of Ect. shaposhnikovii and Chr. minutissium it was found that the characteristic time of cytochrome oxidation, tau, in samples with a high degree of humidity (P/Ps = 0.75) is 2-3 mus, which is close to that seen under physiological conditions (a suspension of intact cells or chromatophores). It was found that under continuous or pulsed illumination the lowering of the relative humidity from 0.75 to 0.15 P/Ps results in a reversible decrease in the amount of cytochrome molecules that can undergo photooxidation. The fraction of cytochrome pool that retains its activity shows a rate of oxidation which remains almost without change. The observed hydration effect and its involvement in the control of the photo-induced oxidation of cytochromes must be taken into account when a model of the molecular mechanism of this process is constructed on the basis of the electron tunneling theory. It is also shown that the dark-reduction kinetics of high-potential cytochrome consist of two components: a fast component with t1/2 = 1-3s which is independent of the sample humidity and a slow component with t1/2 = 5-20 s whose lifetime increases by a factor of 3-5 on reducing the humidity. At a high degree of humidity (P/Ps = 0.75-0.5), the kinetics of cytochrome dark-reduction exhibits only the slow component. The fast component is probably associated with the return of an electron from the primary ferroquinone acceptor and the slow component seems likely to be due to the direct transfer of an electron from exogenous electron donor to the cytochrome.

[Kinetics of light-induced redox changes of high-potential cytochrome in the chromatophores of purple sulfur bacteria Ectothiorhodospira shaposhnikovii]. / Kinetika fotoindutsirovannykh redoks-prevrashchenii vysokopotentsial'nogo tsitokhroma v khromatoforakh sernykh purpurnykh bakterii Ectothiorhodospira shaposhnikovii.

Light-induced redox changes of high-potential cytochrome Ch (Em 7.0 = + 290 mV) have been studied. It was found that after switching off the actinic light there is a delay in the cytochrome dark reduction. The extent of the delay depends on the intensity of actinic light, being the more the higher the intensity. A simple kinetic model is proposed to explain both kinetics of redox changes of the cytochrome and dependence of the delay upon the intensity of actinic light.