Stephen K. Roberts

A patch clamp study of Na + transport in maize roots

The mechanisms mediating Na(+) transpdrt in higher plant roots were investigated by applying the patch clamp technique to protoplasts isolated from the cortex and stele of maize roots. In the cortex, permeation of Na+ through a time-dependent K(+)-selective inward rectifier was negligible. Instead, Na(+) influx into maize roots probably occurs via an instantaneously-activating current. This current was partially inhibited by extracellular Ca(2+), but was insensitive to extracellular TEA(+), Cs(+) and TTX. In outside-out patches, a plasma membrane ion channel was found which mediated an inward Na(+) current which, at least in part, underlies the whole-cell instantaneously-activating current. The unitary conductance of this channel was 15 pS in 102:121 mM Na(+) (outsidexytosol). Channel gating was voltage-independent and distinct from that observed for the inwardly rectifying K(+)-selective channel in the same cell type. Increasing extracellular Ca(2+) from 0.1 to 1 mM reduced the open probability and unitary conductance of this channel. In 102 mM Na(+) : 123 mM K(+) (outside:cytosol) a P(Na):P(K) of 2.1 was calculated. It is suggested that the plasma membrane Na(+)-permeable channel identified in the cortex of maize roots represents a pathway for low affinity Na(+) uptake by intact maize roots. In the stele, permeation of Na(+) through outwardly rectifying K(+) channels was found to be negligible and the channels are thus unlikely to be involved in the transport of Na(+) from the root symplasm.

Characterization of anion channels in the plasma membrane of Arabidopsis epidermal root cells and the identification of a citrate-permeable channel induced by phosphate starvation.

Organic-acid secretion from higher plant roots into the rhizosphere plays an important role in nutrient acquisition and metal detoxification. In this study we report the electrophysiological characterization of anion channels in Arabidopsis (Arabidopsis thaliana) root epidermal cells and show that anion channels represent a pathway for citrate efflux to the soil solution. Plants were grown in nutrient-replete conditions and the patch clamp technique was applied to protoplasts isolated from the root epidermal cells of the elongation zone and young root hairs. Using SO42− as the dominant anion in the pipette, voltage-dependent whole-cell inward currents were activated at membrane potentials positive of −180 mV exhibiting a maximum peak inward current (Ipeak) at approximately −130 mV. These currents reversed at potentials close to the equilibrium potential for SO42−, indicating that the inward currents represented SO42− efflux. Replacing intracellular SO42− with Cl− or NO3− resulted in inward currents exhibiting similar properties to the SO42− efflux currents, suggesting that these channels were also permeable to a range of inorganic anions; however when intracellular SO42− was replaced with citrate or malate, no inward currents were ever observed. Outside-out patches were used to characterize a 12.4-picoSiemens channel responsible for these whole-cell currents. Citrate efflux from Arabidopsis roots is induced by phosphate starvation. Thus, we investigated anion channel activity from root epidermal protoplasts isolated from Arabidopsis plants deprived of phosphate for up to 7 d after being grown for 10 d on phosphate-replete media (1.25 mm). In contrast to phosphate-replete plants, protoplasts from phosphate-starved roots exhibited depolarization-activated voltage-dependent citrate and malate efflux currents. Furthermore, phosphate starvation did not regulate inorganic anion efflux, suggesting that citrate efflux is probably mediated by novel anion channel activity, which could have a role in phosphate acquisition.

The Saccharomyces cerevisiae Ca2+ channel Cch1pMid1p is essential for tolerance to cold stress and iron toxicity.

Cch1p and Mid1p are components of a high‐affinity Ca2+‐permeable channel in the yeast plasma membrane. Here, we show that growth of mutants in the Cch1pMid1p channel is markedly hypersensitive to low temperature and to high iron concentration in the medium. Both phenotypes were suppressed by high Ca2+ concentration. Iron stress elicited an increased Ca2+ influx into both wild type and cch1Δmid1Δ yeast. Inhibition of calcineurin strongly depressed growth of iron‐stressed wild type yeast, indicating that calcineurin is a downstream element of the iron stress response. Iron hypersensitivity of the cch1Δmid1Δ mutant was not associated with an increased iron uptake. An involvement of oxidative stress in the iron‐hypersensitive phenotype was indicated by the findings that the antioxidants tocopheryl acetate and (ethyl)glutathione improved growth and viability of the iron‐stressed mutant. Further, the degree of glutathione oxidation was increased in the presence of iron. The results indicate that iron stress leads to an increased oxidative poise and that Cch1pMid1p is essential to tolerate this condition.

Cch1p Mediates Ca2+ Influx to Protect Saccharomyces cerevisiae against Eugenol Toxicity

Eugenol has antifungal activity and is recognised as having therapeutic potential. However, little is known of the cellular basis of its antifungal activity and a better understanding of eugenol tolerance should lead to better exploitation of eugenol in antifungal therapies. The model yeast, Saccharomyces cerevisiae, expressing apoaequorin was used to show that eugenol induces cytosolic Ca2+ elevations. We investigated the eugenol Ca2+ signature in further detail and show that exponentially growing cells exhibit Ca2+ elevation resulting exclusively from the influx of Ca2+ across the plasma membrane whereas in stationary growth phase cells Ca2+ influx from intracellular and extracellular sources contribute to the eugenol-induced Ca2+ elevation. Ca2+ channel deletion yeast mutants were used to identify the pathways mediating Ca2+ influx; intracellular Ca2+ release was mediated by the vacuolar Ca2+ channel, Yvc1p, whereas the Ca2+ influx across the plasma membrane could be resolved into Cch1p-dependent and Cch1p-independent pathways. We show that the growth of yeast devoid the plasma membrane Ca2+ channel, Cch1p, was hypersensitive to eugenol and that this correlated with reduced Ca2+ elevations. Taken together, these results indicate that a cch1p-mediated Ca2+ influx is part of an intracellular signal which protects against eugenol toxicity. This study provides fresh insight into the mechanisms employed by fungi to tolerate eugenol toxicity which should lead to better exploitation of eugenol in antifungal therapies.

Calcium Dependence of Eugenol Tolerance and Toxicity in Saccharomyces cerevisiae

Eugenol is a plant-derived phenolic compound which has recognised therapeutical potential as an antifungal agent. However little is known of either its fungicidal activity or the mechanisms employed by fungi to tolerate eugenol toxicity. A better exploitation of eugenol as a therapeutic agent will therefore depend on addressing this knowledge gap. Eugenol initiates increases in cytosolic Ca2+ in Saccharomyces cerevisiae which is partly dependent on the plasma membrane calcium channel, Cch1p. However, it is unclear whether a toxic cytosolic Ca2+elevation mediates the fungicidal activity of eugenol. In the present study, no significant difference in yeast survival was observed following transient eugenol treatment in the presence or absence of extracellular Ca2+. Furthermore, using yeast expressing apoaequorin to report cytosolic Ca2+ and a range of eugenol derivatives, antifungal activity did not appear to be coupled to Ca2+ influx or cytosolic Ca2+ elevation. Taken together, these results suggest that eugenol toxicity is not dependent on a toxic influx of Ca2+. In contrast, careful control of extracellular Ca2+ (using EGTA or BAPTA) revealed that tolerance of yeast to eugenol depended on Ca2+ influx via Cch1p. These findings expose significant differences between the antifungal activity of eugenol and that of azoles, amiodarone and carvacrol. This study highlights the potential to use eugenol in combination with other antifungal agents that exhibit differing modes of action as antifungal agents to combat drug resistant infections.

Sodium binding sites and permeation mechanism in the NaChBac channel : a molecular dynamics study

NaChBac was the first discovered bacterial sodium voltage-dependent channel, yet computational studies are still limited due to the lack of a crystal structure. In this work, a pore-only construct built using the NavMs template was investigated using unbiased molecular dynamics and metadynamics. The potential of mean force (PMF) from the unbiased run features four minima, three of which correspond to sites IN, CEN, and HFS discovered in NavAb. During the run, the selectivity filter (SF) is spontaneously occupied by two ions, and frequent access of a third one is often observed. In the innermost sites IN and CEN, Na+ is fully hydrated by six water molecules and occupies an on-axis position. In site HFS sodium interacts with a glutamate and a serine from the same subunit and is forced to adopt an off-axis placement. Metadynamics simulations biasing one and two ions show an energy barrier in the SF that prevents single-ion permeation. An analysis of the permeation mechanism was performed both computing minimum energy paths in the axial-axial PMF and through a combination of Markov state modeling and transition path theory. Both approaches reveal a knock-on mechanism involving at least two but possibly three ions. The currents predicted from the unbiased simulation using linear response theory are in excellent agreement with single-channel patch-clamp recordings.

Characterisation of AnBEST1, a functional anion channel in the plasma membrane of the filamentous fungus, Aspergillus nidulans.

Two distant homologues of the bestrophin gene family have been identified in the filamentous fungus, Aspergillus nidulans (anbest1 and anbest2). AnBEST1 was functionally characterised using the patch clamp technique and was shown to be an anion selective channel permeable to citrate. Furthermore, AnBEST1 restored the growth of the pdr12Δ yeast mutant on inhibitory concentrations of extracellular propionate, benzoate and sorbate, also consistent with carboxylated organic anion permeation of AnBEST1. Similar to its animal counterparts, AnBEST1 currents were activated by elevated cytosolic Ca(2+) with a K(d) of 1.60μM. Single channel currents showed long (>10s) open and closed times with a unitary conductance of 16.3pS. Transformation of A. nidulans with GFP-tagged AnBEST1 revealed that AnBEST1 localised to the plasma membrane. An anbest1 null mutant was generated to investigate the possibility that AnBEST1 mediated organic anion efflux across the plasma membrane. Although organic anion efflux was reduced from anbest1 null mutants, this phenotype was linked to the restoration of uracil/uridine-requiring A. nidulans strains to uracil/uridine prototrophy. In conclusion, this study identifies a new family of organic anion-permeable channels in filamentous fungi. We also reveal that uracil/uridine-requiring Aspergillus strains exhibit altered organic anion metabolism which could have implications for the interpretation of physiological studies using auxotrophic Aspergillus strains.

Valproate inhibits MAP kinase signalling and cell cycle progression in S. cerevisiae

The mechanism of action of valproate (VPA), a widely prescribed short chain fatty acid with anticonvulsant and anticancer properties, remains poorly understood. Here, the yeast Saccharomyces cerevisiae was used as model to investigate the biological consequences of VPA exposure. We found that low pH strongly potentiates VPA-induced growth inhibition. Transcriptional profiling revealed that under these conditions, VPA modulates the expression of genes involved in diverse cellular processes including protein folding, cell wall organisation, sexual reproduction, and cell cycle progression. We further investigated the impact of VPA on selected processes and found that this drug: i) activates markers of the unfolded protein stress response such as Hac1 mRNA splicing; ii) modulates the cell wall integrity pathway by inhibiting the activation of the Slt2 MAP kinase, and synergizes with cell wall stressors such as micafungin and calcofluor white in preventing yeast growth; iii) prevents activation of the Kss1 and Fus3 MAP kinases of the mating pheromone pathway, which in turn abolishes cellular responses to alpha factor; and iv) blocks cell cycle progression and DNA replication. Overall, our data identify heretofore unknown biological responses to VPA in budding yeast, and highlight the broad spectrum of cellular pathways influenced by this chemical in eukaryotes.

Calcium Imaging of the Cyclic Nucleotide Response

Calcium (Ca(2+)) is a key component of the signalling network by which plant cells respond to developmental and environmental signals. A change in guard cell cytosolic free Ca(2+)([Ca(2+)]cyt) is an early event in the response of stomata to both opening and closing stimuli, and cyclic nucleotide-mediated Ca(2+) signalling has been implicated in the regulation of stomatal aperture. A range of techniques have been used to measure [Ca(2+)]cyt in plant cells. Here we describe a potential method for imaging cyclic nucleotide-induced changes in [Ca(2+)]cyt in guard cells using the cameleon ratiometric Ca(2+) reporter protein.

Spectral Decomposition of Geodesic Flows on Constant Curvature Surfaces

The dynamics of a Hamiltonian system can either be described in terms of the trajectories \( x_{\rm{0}} \to x\left( t \right) = U_t x_{\rm{0}} \) in the phase space, or by specifying the laws of evolution of a function \( \phi \left( x \right) \) on the phase space:\( \phi \to \hat U_t \phi \) . The evolution operator \( \hat U_t \) which pulls the function backwards along the trajectories is defined by