Jesús Muñiz

Inhibition of Vacuolar Ion Channels by Polyamines

Abstract. 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.

Conduction of Monovalent and Divalent Cations in the Slow Vacuolar Channel

Abstract. 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 ≤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 (∼140 Å) 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.

Mechanism of luminal Ca2+ and Mg2+ action on the vacuolar slowly activating channels

The non-selective slow vacuolar (SV) channel can dominate tonoplast conductance, making it necessary to tightly control its activity. Applying the patch-clamp technique to vacuoles from sugar beet (Beta vulgaris L.) taproots we studied the effect of divalent cations on the vacuolar side of the SV channel. Our results show that the SV channel has two independent binding sites for vacuolar divalent cations, (i) a less selective one, inside the channel pore, binding to which impedes channel conductance, and (ii) a Ca2+-selective one outside the membrane-spanning part of the channel protein, binding to which stabilizes the channel’s closed conformations. Vacuolar Ca2+ and Mg2+ almost indiscriminately blocked ion fluxes through the open channel pore, decreasing measured single-channel current amplitudes. This low-affinity block displays marked voltage dependence, characteristic of a ‘permeable blocker’. Vacuolar Ca2+—with a much higher affinity than Mg2+—slows down SV channel activation and shifts the voltage dependence to more (cytosol) positive potentials. A quantitative analysis results in a model that exactly describes the Ca2+-specific effects on the SV channel activation kinetics and voltage gating. According to this model, multiple (approximately three) divalent cations bind with a high affinity at the luminal interface of the membrane to the channel protein, favoring the occupancy of one of the SV channel’s closed states (C2). Transition to another closed state (C1) diminishes the effective number of bound cations, probably due to mutual repulsion, and channel opening is accompanied by a decrease of binding affinity. Hence, the open state (O) is destabilized with respect to the two closed states, C1 and C2, in the presence of Ca2+ at the vacuolar side. The specificity for Ca2+ compared to Mg2+ is explained in terms of different binding affinities for these cations. In this study we demonstrate that vacuolar Ca2+ is a crucial regulator to restrict SV channel activity to a physiologically meaningful range, which is less than 0.1% of maximum SV channel activity.

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

Abstract 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 (zδ 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.

Homeostatic control of slow vacuolar channels by luminal cations and evaluation of the channel-mediated tonoplast Ca2+ fluxes in situ

Ca2+, Mg2+, and K+ activities in red beet (Beta vulgaris L.) vacuoles were evaluated using conventional ion-selective microelectrodes and, in the case of Ca2+, by non-invasive ion flux measurements (MIFE) as well. The mean vacuolar Ca2+ activity was ∼0.2 mM. Modulation of the slow vacuolar (SV) channel voltage dependence by Ca2+ in the absence and presence of other cations at their physiological concentrations was studied by patch-clamp in excised tonoplast patches. Lowering pH at the vacuolar side from 7.5 to 5.5 (at zero vacuolar Ca2+) did not affect the channel voltage dependence, but abolished sensitivity to luminal Ca2+ within a physiological range of concentrations (0.1–1.0 mM). Aggregation of the physiological vacuolar Na+ (60 mM) and Mg2+ (8 mM) concentrations also results in the SV channel becoming almost insensitive to vacuolar Ca2+ variation in a range from nanomoles to 0.1 mM. At physiological cation concentrations at the vacuolar side, cytosolic Ca2+ activates the SV channel in a voltage-independent manner with Kd=0.7–1.5 μM. Comparison of the vacuolar Ca2+ fluxes measured by both the MIFE technique and from estimating the SV channel activity in attached patches, suggests that, at resting membrane potentials, even at elevated (20 μM) cytosolic Ca2+, only 0.5% of SV channels are open. This mediates a Ca2+ release of only a few pA per vacuole (∼0.1 pA per single SV channel). Overall, our data suggest that the release of Ca2+ through SV channels makes little contribution to a global cytosolic Ca2+ signal.

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.

Effects of external calcium on potassium contractures in tonic muscle fibers of the frog (Rana pipiens).

K+ contractures of tonic bundles from cruralis muscle of the frog were studied with different K+ concentrations (10‐120 mM). K+ contractures had an initial transient phase followed by a sustained tension. The amplitude of the sustained tension diminished with high K+ concentration (80‐120 mM). However, in all cases, tension was maintained for several minutes. External Ca2+ reduction practically abolished the sustained phase of the K+ contractures. The initial phase was also reduced and tension spontaneously relaxed. The curve relating the peak tension with log [K+]o, showed that the threshold was not affected but the peak tension was reduced to about 70% in low‐Ca2+ saline (0 Ca2+ + 3 mM‐Mg2+) and 50% in Ca2+‐free saline (1 mM‐EGTA + 3 mM‐Mg2+). The dependence of the sustained tension on external Ca2+ was further confirmed by Ca2+ withdrawal and re‐establishment and/or by Ni2+ substitution for Ca2+ before or during K+ contractures. These results indicate that external Ca2+ had to be continuously present to maintain the tension during K+ contractures and that Ni2+ was not able to restore the normal temporal course of K+ contracture. The sustained phase was diminished by blocking agents of Ca2+ channels, such as nifedipine (1 microM) and diltiazem (1‐10 microM). The present results can be explained by a direct control of the Ca2+ currents on K+ contracture or by specific interactions between external Ca2+ and Ca2+‐binding sites in the membrane.

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

A prerequisite for photosynthetic CO2 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 ∼40 pS to ∼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+, Ca2+, Mg2+) 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 Gd3+. 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.

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 Ca2+-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−/nm2. Negative surface potential also tended to increase the local concentration of permeable ions (K+), resulting in anomalously high single-channel conductance, ∼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 Ca2+, 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 Ca2+ in the membrane vicinity, thus reducing the inhibitory effect of vacuolar Ca2+ and causing a negative shift of the SV channel voltage dependence, with a sum of the two shifts being negative.

Passive mechanical properties of cardiac tissues in heart hypertrophy during pregnancy

We evaluated changes in passive mechanical properties in cardiac tissues during rat pregnancy. Left and right ventricular free walls were dissected from hearts of nonpregnant, late-pregnant, and postpartum rats. Mechanical experiments in ventricular strips were done by stretch–release cycles using a step motor. The results show that during pregnancy, there is cardiac hypertrophy associated with (1) an increase in myocyte size, particularly of augmented myocyte length, (2) a decrease in passive tension developed by the myocardial walls, and (3) a decrease in both elastic modulus and hysteresis. All changes observed during rat pregnancy were reversed during postpartum. In conclusion, a heart with less ventricular rigidity could contribute to facilitating the ventricular filling in conditions of a greater circulating volume characteristic of pregnancy.