Monica Bregante

Impaired pH Homeostasis in Arabidopsis Lacking the Vacuolar Dicarboxylate Transporter and Analysis of Carboxylic Acid Transport across the Tonoplast

Arabidopsis (Arabidopsis thaliana) mutants lacking the tonoplastic malate transporter AttDT (A. thaliana tonoplast dicarboxylate transporter) and wild-type plants showed no phenotypic differences when grown under standard conditions. To identify putative metabolic changes in AttDT knock-out plants, we provoked a metabolic scenario connected to an increased consumption of dicarboxylates. Acidification of leaf discs stimulated dicarboxylate consumption and led to extremely low levels of dicarboxylates in mutants. To investigate whether reduced dicarboxylate concentrations in mutant leaf cells and, hence, reduced capacity to produce OH− to overcome acidification might affect metabolism, we measured photosynthetic oxygen evolution under conditions where the cytosol is acidified. AttDT::tDNA protoplasts showed a much stronger inhibition of oxygen evolution at low pH values when compared to wild-type protoplasts. Apparently citrate, which is present in higher amounts in knock-out plants, is not able to replace dicarboxylates to overcome acidification. To raise more information on the cellular level, we performed localization studies of carboxylates. Although the total pool of carboxylates in mutant vacuoles was nearly unaltered, these organelles contained a lower proportion of malate and fumarate and a higher proportion of citrate when compared to wild-type vacuoles. These alterations concur with the observation that radioactively labeled malate and citrate are transported into Arabidopsis vacuoles by different carriers. In addition, wild-type vacuoles and corresponding organelles from AttDT::tDNA mutants exhibited similar malate channel activities. In conclusion, these results show that Arabidopsis vacuoles contain at least two transporters and a channel for dicarboxylates and citrate and that the activity of AttDT is critical for regulation of pH homeostasis.

The voltage-dependent potassium-uptake channel of corn coleoptiles has permeation properties different from other K(+)channels

The initial response of coleoptile cells to growth hormones and light is a rapid change in plasma-membrane polarization. We have isolated protoplasts from the cortex of maize (Zea mays L.) coleoptiles to study the electrical properties of their plasma membrane by the patch-clamp techniqueUsing the whole-cell configuration and cell-free membrane patches we could identify an H+-ATPase, hyperpolarizing the membrane potential often more negative than -150 mV, and a voltage-dependent, inward-rectifying K+ channel (unit conductance ≈5–7 pS) as the major membrane conductan-ces Potassium currents through this channel named CKC1in (for Coleoptile K+ Channel inward rectifier) were elicited upon voltage steps negative to -80 mV, characterized by a half-activation potential of -112 mV. The kinetics of activation, well described by a double-exponential process, were strongly dependent on the degree of hyperpolarization and the cytoplasmic Ca2+ level. Whereas at nanomolar Ca2+ concentrations K+ currents increased with a t1/2=16 ms (at -180 mV), higher calcium levels slowed the activation process about fourto fivefoldUpon changes in the extracellular K+ concentration the reversal potential of the K+ channel followed the Nernst potential for potassium with a 56-mV shift for a tenfold increaseThe absence of a measurable conductance for Na+, Rb+, Cs+ and a permeability ratio PNH4+/PK+ around 0.25 underlines the high selectivity of CKC1in for K+In contrast to Cs+, which at submillimolar concentration blocks the channel in a voltage-dependent manner, Rb+, often used as a tracer for K+, does not permeate this type of K+ channelThe lack of Rb+ permeability is unique with respect to other K+ transporters. Therefore, future molecular analysis of CKC1in, considered as a unique variation of plant inward rectifiers, might help to understand the permeation properties of K+ channels in general.

Single mutations strongly alter the K+-selective pore of the Kin channel KAT1

Voltage‐dependent potassium uptake channels represent the major pathway for K+ accumulation underlying guard cell swelling and stomatal opening. The core structure of these Shaker‐like channels is represented by six transmembrane domains and an amphiphilic pore‐forming region between the fifth and sixth domain. To explore the effect of point mutations within the stretch of amino acids lining the K+ conducting pore of KAT1, an Arabidopsis thaliana guard cell Kin channel, we selected residues deep inside and in the periphery of the pore. The mutations on positions 256 and 267 strongly altered the interaction of the permeation pathway with external Ca2+ ions. Point mutations on position 256 in KAT1 affected the affinity towards Ca2+, the voltage dependence as well as kinetics of the Ca2+ blocking reaction. Among these T256S showed a Ca2+ phenotype reminiscent of an inactivation‐like process, a phenomenon unknown for Kin channels so far. Mutating histidine 267 to alanine, a substitution strongly affecting C‐type inactivation in Shaker, this apparent inactivation could be linked to a very slow calcium block. The mutation H267A did not affect gating but hastened the Ca2+ block/unblock kinetics and increased the Ca2+ affinity of KAT1. From the analysis of the presented data we conclude that even moderate point mutations in the pore of KAT1 seem to affect the pore geometry rather than channel gating.

DKT1, a novel K+ channel from carrot, forms functional heteromeric channels with KDC1

We report the isolation and characterisation of DKT1, a new carrot K+ channel α‐subunit belonging to the Shaker‐like family. DKT1 is expressed in many tissues of the adult plant, suggesting that it may play important roles in both nutrition and other important physiological processes. During embryo development, DKT1 is expressed at later phases implying the involvement of K+ in embryo maturation. When co‐expressed with KDC1 in Xenopus oocytes, DKT1 is able to form functional, heteromeric channels, suggesting that possible interactions between these two ion channels in plant tissues may modulate K+ uptake.

Permeability Increase Induced by Escherichia coli Hemolysin A in Human Macrophages is Due to the Formation of Ionic Pores: A Patch Clamp Characterization

Abstract.Escherichia coli hemolysin is known to cause hemolysis of red blood cells by forming hydrophilic pores in their cell membrane. Hemolysin-induced pores have been directly visualized in model systems such as planar lipid membranes and unilamellar vesicles. However this hemolysin, like all the members of a related family of toxins called Repeat Toxins, is a potent leukotoxin. To investigate whether the formation of channels is involved also in its leukotoxic activity, we used patch-clamped human macrophages as targets. Indeed, when exposed to the hemolysin, these cells developed additional pores into their membrane. Such exogenous pores had properties very different from the endogenous channels already present in the cell membrane (primarily K+ channels), but very similar to the pores formed by the toxin in purely lipidic model membranes. Observed properties were: large single channel conductance, cation over anion selectivity but weak discrimination among different cations, quasilinear current-voltage characteristic and the existence of a flickering pre-open state of small conductance. The selectivity properties of the toxin channels appearing in phospholipid vesicles were also investigated, using a specially adapted polarization/depolarization assay, and were found to be completely consistent with that of the current fluctuations observed in excised macrophage patches.

KDC1, a carrot Shaker-like potassium channel, reveals its role as a silent regulatory subunit when expressed in plant cells.

The Shaker potassium channels are tetrameric proteins formed by the assembly of four α-subunits. The oligomerization can occur among both homo- and hetero-α-subunits. KDC1 is a carrot Shaker-like potassium channel expressed in the epidermis of plantlet roots and the protoderm of somatic embryos. KDC1 was previously characterised electrophysiologically in CHO and Xenopus oocytes cells, but the experiments performed in these systems did not provide conclusive evidence that KDC1 forms a functional homomeric channel in plant cells. In this report, we show that KDC1 localizes to the plasma membrane of root cells in transgenic tobacco plants transformed with a KDC1∷GFP fusion construct. In tobacco mesophyll protoplasts, transiently transformed with KDC1∷GFP, KDC1 was present on the endomembrane and the protoplasts did not show any inward potassium current, as demonstrated by patch-clamp experiments. The co-expression of KDC1∷GFP with the Arabidopsisthaliana potassium channel AKT1 in tobacco mesophyll protoplasts has the effect of shifting KDC1 localization from endomembranes to the plasma membrane. Patch-clamp experiments performed on tobacco mesophyll protoplasts expressing AKT1 alone or in combination with KDC1∷GFP showed voltage-activated inward potassium currents with different properties. In particular, the addition of Zn2+ to the bath solution induced a clear decrease of the potassium currents in protoplasts transformed with AKT1 alone, whereas a current potentiation (indicative of KDC1 presence) was observed in protoplasts co-transformed with AKT1 + KDC1∷GFP. Split-Ubiquitin assay experiments performed in yeast cells confirmed the interaction between AKT1 and KDC1.

Histidines are responsible for zinc potentiation of the current in KDC1 carrot channels.

Unlike all plant inward-rectifying potassium channels, the carrot channel KDC1 has two histidine pairs (H161,H162) in the S3-S4 and (H224,H225) in the S5-S6 linkers. When coinjected with KAT1 in Xenopus oocytes, KDC1 participates in the formation of heteromultimeric KDC1:KAT1 channels and the ionic current is potentiated by extracellular Zn2+. To investigate the potential interactions between KDC1 and zinc, a KDC1-KAT1 dimer was constructed. The dimeric and heteromeric channels displayed similar characteristics and the same sensitivity to zinc and other metals; this result suggests that zinc binding is mediated by residues in a single channel subunit. The KDC1:KAT1 currents were also potentiated by external Pb2+ and Cd2+ and inhibited by Ni2+. To investigate further the role of KDC1-histidines, these amino acids were mutated into alanines. The single mutations H225A, H161A, and H162A did not affect the response of the heteromeric channels to zinc. Conversely, the single mutant H224A and the double mutants (H224A,H225A) and (H161A,H162A) abolished zinc potentiation, but not that induced by Pb2+ or Cd2+. These results suggest that Zn2+ potentiation cannot be ascribed to simple electrostatic interactions between zinc and channel residues and that histidine 224 is crucial for zinc but not for lead potentiation of the current.

A novel K+ channel expressed in carrot roots with a low susceptibility toward metal ions.

Kdc1 is a novel K+-channel gene cloned from carrot roots, and which is also present in cultured carrot cells. We investigated the characteristics of the ionic current elicited in Xenopus oocytes coinjected with KDC1 (K+-Daucus carota 1) and KAT1 (from Arabidopsis thaliana) RNA. Expressed heteromeric channels displayed inward-rectifying potassium currents whose kinetics, voltage characteristics, and inhibition by metal ions depended on KDC1:KAT1 ratios. At low KDC1:KAT1 ratios, Zn2+ inhibition of heteromeric K+ current was less pronounced compared to homomeric KAT1 channels, while at higher KDC1:KAT1 ratios, the addition of Zn2+ even produced an increase in current. Under the same conditions, the Ni2+ inhibition of the current was also reduced, but no current increase was observed. These effects might be explained by the unusual amino acid composition of the KDC1 protein in terms of histidine residues that are absent in the pore region, but abundant (four per subunit) in the proximity of the pore entrance. Channels like KDC1 could be at least partially responsible for the higher resistance of carrot cells in the presence of metals.

BACTERIAL HEMOLYSINS AND LEUKOTOXINS AFFECT TARGET CELLS BY FORMING LARGE EXOGENOUS PORES INTO THEIR PLASMA MEMBRANE. ESCHERICHIA COLI HEMOLYSIN A AS A CASE EXAMPLE

Many bacteria include among their virulence factors exoproteins which exert leukocidal and cytolytic functions and have the ability to form pores in model membranes. We show that, at least in the case of the RTX hemolysin produced byEscherichia coli (HlyA), formation of pores in planar lipid membranes is parallelled by opening of strikingly similar channels in the plasma membrane of exposed macrophages. Formation of such lesions in leukocytes can give rise to a variety of effects leading altogether to a diminished immune response towards the invasive bacteria.

Effects of mono- and multi-valent cations on the inward-rectifying potassium channel in isolated protoplasts from maize roots

Abstract Transport properties mediated by ionic channels were studied by the patch-clamp technique in protoplasts from cortical parenchyma cells of maize roots (CPMR). While outward currents could be seen only occasionally, macroscopic voltage- and time-dependent potassium-selective inward currents (IK+in) were frequently observed in the whole-cell configuration. These currents increased continuously as a function of K+ concentration (in the range 3 – 200 mm) and the slow-saturating macroscopic chord-conductance was fitted by a Michaelis-Menten function with Km = 195 ± 39 mm. Other ions, like sodium and lithium, did not permeate at all through the maize root inward-channel, or like ammonium (PNH4+/ PK+ = 0.16 0.25) and rubidium (PRb+/PK+≈ 0.10) displayed a very low permeability ratio. Up to 5 mm Rb+ did not induce any inhibition of the K+ inward current, whereas submillimolar concentrations of Cs+ were sufficient to block, in a voltage-dependent manner, the inward currents. A decrease of the external potassium concentration favoured Cs+ inhibition (Km = 89 ± 6 µm and 26 ± 2 µm in 200 and 100 mm KCl, respectively). The potassium inward-currents were reversibly and consistently inhibited by submillimolar external concentrations of the metal ions Ni2+, Zn2+ and Co2+, while 1 mm La3+ only slightly decreased (≈10%) both the single channel conductance (9.2 ± 1.2 pS in 100 mm potassium) and the macroscopic current. In contrast to the case with Cs+, inhibition induced by other metal ions did not show any voltage dependence. These results suggest that, as with animal potassium channels, the inward channel of maize-root cortical cells has a narrow pore of permeation and metal ions decrease the K+ current, possibly by acting on binding sites located outside the pore.