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D Sanders

Publications and source records attributed to D Sanders.

At least 91 records · Page 5Linked to original sources
PubMed1996

Interaction of nisin with planar lipid bilayers monitored by fluorescence recovery after photobleaching.

Nisin, a prominent member of the lantibiotic family of antimicrobial agents, has wide application as a food preservative despite poor understanding of its mode of action. Fluorescence recovery after photobleaching has been used with planar lipid bilayers as a model membrane system to examine how nisin might interact with the surface of bacterial cells. Nisin associates with planar lipid bilayers in the absence of an applied membrane potential causing an array of effects consistent with adsorption of nisin onto the membrane surface which involves inhibition of the lateral diffusion and fluorescence of the lipid probe N-(7--1,2,3-benzoxadiazol-4-yl) phosphatidylethanolamine (NBD-PE) and a reduction of the capacitance of the bilayer. Nisin adsorption is dependent on phospholipid composition. In the presence of dioleoylphosphatidylcholine (PC): cardiolipin (CL) 4:1, the rate of lateral mobility of phospholipid is reduced to 61% of the control level which decreases to a value of 46% when CL is replaced by 1-palmitoyl-2-oleoylphosphatidylserine (PS). These effects on bilayer parameters are transient, and with time the values return to near original levels. High electrical conductivity is observed on application of a voltage ramp suggesting that insertion into the membrane follows surface association. Results have been interpreted in terms of a model in which nisin initially binds to the surface of the membrane causing a modulation of bilayer properties.

Anti-Bacterial Agents↗
PubMed1996

Characterization of csi52, a Cs+ resistant mutant of Arabidopsis thaliana altered in K+ transport.

Plant roots accumulate potassium from a wide range of soil concentrations, utilizing at least two distinct plasma membrane uptake systems with different affinities for the cation. Although details on the structure and function of these transporters are beginning to emerge many prominent questions remain concerning how these proteins function in plants. Such questions can be addressed through the use of well-defined transport mutants. Csi52, a caesium-insensitive mutant of Arabidopsis thaliana which is defective in potassium transport, is further characterized here using conventional electrophysiology, patch-clamp and radiometric approaches to identify the nature of the potassium transport lesion. Rb+ uptake experiments reveal a reduced uptake in csi52 in both the high-and low-affinity uptake range. Patch-clamp analysis indicates that the activity of the predominant inward rectifying channel observed in wild-type cells is extremely low in root protoplasts isolated from csi52, whereas outward rectifying channel activity is comparable between wildtype and mutant. Rb+ uptake studies show that in both wild-type and csi52 the high-affinity uptake pathway is considerably less sensitive to Cs+ than the low-affinity pathway with K1/2 values for Cs+ of around 1.3 and 0.2 mM, respectively. Furthermore, K+ starvation leads to a larger relative increase in high-affinity K+ uptake in the mutant than the wild-type. The results demonstrate the Cs+ sensitivity of each individual uptake pathway is comparable in wild-type and csi52 but the high-affinity pathway is less Cs+ sensitive (in both wild-type and csi52). Therefore, the larger shift toward high-affinity uptake in the mutant compared with the wild-type under K(+)-starvation conditions will endow the mutant with a higher degree of overall Cs+ resistance. The data supply evidence for the hypothesis that the csi52 mutation lies within a gene that regulates the activity of several potassium transport systems and coordinates their relative contribution to overall root K+ uptake.

Arabidopsis↗
PubMed1996

Control of ionic currents in guard cell vacuoles by cytosolic and luminal calcium.

Activity of vacuolar ion channels can be regulated by the cytosolic free Ca2+ concentration ([Ca2+]cyt). Using the whole-vacuole mode of patch-clamp with Vicia faba guard cell vacuoles, three distinct cation currents were apparent that were differentially regulated by [Ca2+]cyt. At 'zero' to 100 nM [Ca2+]cyt, instantaneous currents typical of Fast Vacuolar (FV) channels were activated. A 10 fold KCl gradient directed out of the vacuole increased FV currents (up to fivefold) at negative potentials compared with the currents in symmetrical KCl. At [Ca2+]cyt higher than 100 nM, instantaneous currents became smaller and voltage-independent (non-rectifying) and were typical of Vacuolar K(+)-selective (VK) channels. These currents were less sensitive to a KCl gradient than were the FV currents, being stimulated less than twofold at negative potentials. Reversal potentials measured in the presence of a KCl gradient indicated a high K+ permeability of both FV and VK currents. At [Ca2+]cyt higher than 600 nM time-dependent currents elicited by positive potentials were typical of Slow Vacuolar (SV) channel activation. When the Ca2+ mole fraction in the cytosolic or luminal solution was varied the reversal potential of SV currents (determined by tail current analysis) passed through maximum or minimum values. The resultant calculated apparent permeability ratios varied with ionic conditions but indicated high Ca2+ and K+ permeabilities. If a Cl- permeability was assumed then the apparent PCa was lower. However, substitution of Cl- by the larger (impermeant) anion gluconate had no effect on the reversal potential of SV tail currents in the presence of Ca2+ and a K+ gradient, demonstrating that the assumption of Cl- permeability of the SV channel is invalid. Single-channel SV currents also decreased with increasing cytosolic Ca2+ mole fraction. These data indicate that the SV channel is highly cation selective, shows characteristics typical of a multi-ion pore and derives ion selectivity by Ca2+ binding. The SV channel currents could also be Mg(2+)-activated and were demonstrated to be Mg(2+)-permeable in the absence of Ca2+. The apparent permeability ratio (PMg:PK) also varied under different ionic conditions. The results indicate not only that FV, VK and SV channels are all present in a single cell type, but also that each is differentially regulated by [Ca2+]cyt. The respective roles of these channels in vacuolar ion release are discussed, and possible conditions are presented in which these channels could be activated by disparate signalling pathways during stomatal closure.

Biological Transport↗
PubMed1995

Multi-frequency EPR evidence for a binuclear CuA center in cytochrome c oxidase: studies with a 63Cu- and 65Cu-enriched, soluble domain of the cytochrome ba3 subunit II from Thermus thermophilus.

We have recorded multi-frequency EPR spectra of 63Cu- and 65Cu-labeled, water-soluble CuA-protein from the cytochrome ba3 of T. thermophilus. The spectrum taken at the highest frequency (34.03 GHz) shows no hyperfine structure and is nominally axial with apparent gz approximately 2.18 and gxy approximately 2.00. The spectrum taken at the lowest frequency (3.93 GHz) shows a rich hyperfine structure. Analyses of the spectra show that the observed splitting arises from an interaction of the unpaired electron with two Cu nuclei and support the notion that CuA is a mixed-valent [Cu(II)/Cu(I)] complex in which the unpaired electronic spin is distributed evenly over the two Cu ions.

Binding Sites↗
PubMed1995

The voltage-gated Ca2+ release channel in the vacuolar membrane of sugar beet resides in two activity states.

The voltage-gated Ca2+ release channel in the vacuolar membrane of sugar beet tap roots resides in two states which show similar reversal potentials in biionic conditions but differ dramatically in their single channel current and open frequency. State I has a unitary Ca2+ conductance of 3.9 +/- 0.5 pS and channel openings are rare at vacuolar resting potentials. After spontaneous and reversible transition into State II the unitary Ca2+ conductance increases three-fold, accompanied by a concomitant rise in channel activity over the physiological voltage range. The 15- to 19-fold increase in total Ca2+ current after the State I to State II transition could play an essential role in channel activation during signal transduction.

Biological Transport↗
PubMed1995

Release of Ca2+ from individual plant vacuoles by both InsP3 and cyclic ADP-ribose.

Calcium mobilization from intracellular pools couples many stimuli to responses in plant cells. Cyclic adenosine 5'-diphosphoribose (cADPR), which interacts with a ryanodine receptor in certain animal cells, was shown to elicit calcium release at the vacuolar membrane of beet storage root. The vacuolar calcium release pathway showed similarities to cADPR-gated calcium release in animal cells, including inhibition by ruthenium red, ryanodine activation, and high affinity for cADPR [Michaelis constant (Km) = 24 +/- 7 nanomolar]. Analysis by patch-clamping demonstrated that the cADPR-gated pathway in beet is voltage-dependent over the physiological range, does not spontaneously desensitize, and is colocalized with an inositol 1,4,5-trisphosphate (InsP3)-gated calcium release pathway in individual vacuoles.

Adenosine Diphosphate Ribose↗
PubMed1995

Inositol trisphosphate-dependent and -independent Ca2+ mobilization pathways at the vacuolar membrane of Candida albicans.

Vacuolar membrane vesicles were isolated from Candida albicans protoplasts, and marker enzyme assays were employed to identify the membranes as vacuolar in origin. The mechanisms of Ca2+ uptake and Ca2+ release at the vacuolar membrane were investigated. Ca2+ accumulation by vacuolar membrane vesicles can be generated via H+/Ca2+ antiport. The inside-acid pH is in turn generated by a vacuolar-type H(+)-ATPase, as demonstrated by the sensitivity of Ca2+ uptake to ionophores and the vacuolar H(+)-ATPase inhibitor bafilomycin A1. Vacuolar membrane vesicles exhibit two Ca2+ release pathways: one induced by inositol 1,4,5-trisphosphate (InsP3) and the other by inside-positive voltage. These two pathways are distinct with respect to the amount of Ca2+ released, the nature of response to successive stimuli, and their respective pharmacological profiles. The InsP3-gated pathway exhibits a K0.5 for InsP3 of 2.4 microM but is not activated by inositol 4,5-bisphosphate or inositol 1,3,4,5-tetrakisphosphate at concentrations up to 50 microM. Ca2+ release by InsP3 is blocked partially by low molecular weight heparin. Ca2+ released by the voltage-sensitive pathway occurs at membrane potentials estimated to be over a physiological range from 0 to 80 mV. The voltage-sensitive Ca2+ release pathway can be blocked by lanthanide ions and organic channel blockers such as ruthenium red and verapamil. Furthermore, the voltage-sensitive Ca2+ release pathway exhibits Ca(2+)-induced Ca2+ release. These findings are discussed in relation to the mechanism of Ca(2+)-mediated cellular signaling in C. albicans and other fungi.

Anti-Bacterial Agents↗
PubMed1995

Contrasting roles in ion transport of two K(+)-channel types in root cells of Arabidopsis thaliana.

Plant roots accumulate K+ over a range of external concentrations. Root cells have evolved at least two parallel plasma-membrane K+ transporters which operate at millimolar and micromolar external [K+]: high-affinity K+ uptake is energised by symport with H+, while low-affinity uptake is assumed to occur via ion channels. To determine the role of ion channels in low-affinity K+ uptake, a characterisation of the principal K(+)-selective ion channels in the plasma membrane of Arabidopsis thaliana (L.) Heynh. cv. Columbia roots was undertaken. Two classes of K(+)-selective channels were frequently observed: one inward (IRC) and one outward (ORC) rectifying with unitary conductances of 5 pS, 20 pS (IRCs) and 15 pS (ORC), measured in symmetrical 10 mM KCl. The dominant IRC (5 pS) and ORC (15 pS) were highly cation-selective (PCl:PK < 0.025) but less selective amongst monovalent cations (PNa:PK approximately 0.17-0.3). Both the IRC and the ORC were blocked by Ba2+, Cs+ and tetra-ethyl-ammonium, whereas 4-aminopyridine and quinidine selectively inhibited the ORC. The ORC open probability was steeply voltage-dependent and ORC activation potentials were close to the potassium equilibrium potential (EK+), enabling ORCs to conduct mainly outward, but occasionally inward, K+ current. By contrast, gating of the 5-pS IRC was weakly voltage-ependent and IRC gating was invariably restricted to membrane potentials more negative than EK+, ensuring K+ transport was always inwardly directed.(ABSTRACT TRUNCATED AT 250 WORDS)

Arabidopsis↗
PubMed1995

ATP, pH and Mg2+ modulate a cation current in Beta vulgaris vacuoles: a possible shunt conductance for the vacuolar H(+)-ATPase.

Macroscopic instantaneous and time-dependent currents have been measured in the vacuolar membrane of Beta vulgaris using a patch clamp configuration analogous to whole cell mode. At low cytosolic Ca2+ and in the absence of Mg2+, only an instantaneous current was observed. This current is carried predominantly by cations (PK:PCl 7:1, PNa:PCl 4:1 and arginine is also conducted). The instantaneous current can be activated by ATP4- (e.g., ATP-activated mean K+ current density was -20 mA.m-2 at a membrane voltage of -20 mV) and by increasing cytosolic pH and Mg2+ (raising Mg2+ from 0 to 0.4 mM induced a mean current density increase of -7 mA.m-2 at -20 mV). Such current can be activated by simultaneous addition of putative in vivo concentrations of ATP4-/MgATP/Mg2+free (in the presence of bafilomycin to inhibit the vacuolar ATPase) and further modulated by cytosolic pH. With vacuolar K+ concentration greater than that of the cytosol, activation of the instantaneous current would mediate vacuolar K+ release over the range of physiological membrane voltage. It is argued that the ATP(4-)-activated current, in addition to acting as a K+ mobilization pathway, could provide a counter-ion (shunt) conductance, allowing the two electrogenic H+ pumps which reside in the vacuolar membrane to acidify the vacuolar lumen. A separate time-dependent current, which was not observed at low Ca2+ concentrations (less than 500 nM) could also be elicited by addition of Mg2+ at the cytoplasmic membrane face. This current was stimulated by increasing cytoplasmic pH.

Adenosine Triphosphate↗
PubMed1995

Lumenal calcium modulates unitary conductance and gating of a plant vacuolar calcium release channel.

The patch clamp technique has been used to investigate ion permeation and Ca(2+)-dependent gating of a voltage-sensitive Ca2+ release channel in the vacuolar membrane of sugar beet tap roots. Reversal potential measurements in bi-ionic conditions revealed a sequence for permeability ratios of Ca2+ approximately Sr2+ approximately Ba2+ > Mg2+ >> K+ which is inversely related to the size of the unitary conductances K+ >> Mg2+ approximately Ba2+ > Sr2+ approximately Ca2+, suggesting that ion movement is not independent. In the presence of Ca2+, the unitary K+ current is reduced in a concentration- and voltage-dependent manner by Ca2+ binding at a high affinity site (K0.5 = 0.29 mM at 0 mV) which is located 9% along the electric field of the membrane from the vacuolar side. Comparison of reversal potentials obtained under strictly bi-ionic conditions with those obtained in the presence of mixtures of the two ions indicates that the channel forms a multi-ion pore. Lumenal Ca2+ also has an effect on voltage-dependent channel gating. Stepwise increases of vacuolar Ca2+ from micromolar to millimolar concentrations resulted in a dramatic increase in channel openings over the physiological voltage range via a shift in threshold for channel activation to less negative membrane potentials. The steepness of the concentration dependence of channel activation by Ca2+ at -41 mV predicts that two Ca2+ ions need to bind to open the gate. The implications of the results for ion permeation and channel gating are discussed.

Calcium↗
PubMed1995

Mechanism of pyrithione-induced membrane depolarization in Neurospora crassa.

Pyrithione is a general inhibitor of membrane transport in fungi and is widely used in antidandruff shampoos as an antifungal agent. An electrophysiological approach has been used to determine the mode of action of pyrithione on the plasma membrane of the model ascomycete, Neurospora crassa. At pH 5.8, pyrithione induces a dramatic dose-dependent electrical depolarization of the membrane which is complete within 4 min, amounts to 110 mV at saturating pyrithione concentrations, and is half maximal between 0.6 and 0.8 mM pyrithione. Zinc pyrithione induces a similar response but exerts a half-maximal effect at around 0.3 mM. The depolarization is strongly dependent on external pH, being almost absent at pH 8.2, at which the concentration of the uncharged form of pyrithione--which might be expected to permeate the membrane freely--is markedly lowered. However, quantitative considerations based on cytosolic buffer capacity, the pKa of pyrithione, and the submillimolar concentration at which it is active appear to preclude significant cytosolic acidification on dissociation of the thiol proton from the uncharged form of pyrithione. Current-voltage analysis demonstrates that the depolarization is accompanied by a decrease in membrane electrical conductance in a manner consistent with inhibition of the primary proton pump and inconsistent with a mode of action of pyrithione on plasma membrane ion channels. We conclude that pyrithione inhibits membrane transport via a direct or indirect effect on the primary proton pump which energizes transport and that the site of action of pyrithione is likely to be intra- rather than extracellular.

Antifungal Agents↗