Uptake of the lipophilic cation dibenzyldimethylammonium into Saccharomyces cerevisiae. Interaction with the thiamine transport system

The distribution ratio of the lipophilic cation dibenzyldimethylammonium between the cells of Saccharomyces cerevisiae and the medium appears to reflect changes in the membrane potential in a way that is qualitatively correct: the addition of a proton conductor or of an agent which blocks metabolism causes an apparent depolarization of the cell membrane; monovalent cations cause also a lowering of the equilibrium distribution, whereas the addition of divalent cations results in an increase of the partition ratio.However, uptake of dibenzyldimethylammonium and probably also of other liophilic cations proceeds via the thiamine transport system of the yeast. Dibenzyldimethylammonium transport is inducible, like thiamine transport. A kinetic analysis of the mutual interaction between thiamine and dibenzyldimethylammonium uptake shows that these compounds share a common transport system; moreover, dibenzyldimethylammonium uptake is inhibited completely by thiamine disulfide, a competitive inhibitor of thiamine transport and dibenzyldimethylammonium uptake in a thiamine-transport mutant is reduced considerably.It is concluded that one should be cautious when using lipophilic cations to measure the membrane potential of cells of S. cerevisiae.     

Measurement of membrane potential inBacillus subtilis: A comparison of lipophilic cations,rubidium ion,and a cyanine dye as probes

Summary Two of the commonly used probes for measuring membrane potential—lipophilic cations and the cyanine dye diS-C₃(5)—indicated nominally opposite results when tetraphenylarsonium ion was added as a drug to suspensions of metabolizingBacillus subtilis cells. [³H]-Triphenylmethylphosphonium uptake was enhanced by the addition, indicating hyperpolarization, yet fluorescence of diS-C₃(5) was also enhanced, indicating depolarization. Evidence is presented that both effects are artifactual, and can occur without any change in membrane potential, as estimated by⁸⁶Rb⁺ uptake in the presence of valinomycin. The fluorescence studies suggest that tetraphenylarsonium ion displaces the cyanine dye from the cell envelope, or other binding site, into the aqueous phase.The uptake characteristics of the radiolabeled lipophilic cations were quite unusual: At low concentrations (e.g., less than 10 m for triphenylmethylphosphonium) there was potential-dependent uptake of the label to a stable level, but subsequent addition of nonradioactive lipophilic cation caused further uptake of label to a new stable level. Labeled triphenylmethylphosphonium ion taken up to the first stable level could be displaced by 10mm magnesium ion, whereas⁸⁶Rb⁺ uptake was unperturbed. Association of the lipophilic cations with the surface of de-energized cells was concentration-dependent, but there was no evidence for cooperative binding. This phenomenon of stimulated uptake inB. subtilis (which was not seen inEscherichia coli cells or vesicles) is consistent with a two-compartment model with access to the second compartment only being possible above a critical cation concentration. We tentatively propose such a model, in which these compartments are the cell surface and the cytoplasm, respectively.Triphenylmethylphosphonium up to 0.5mm exhibited linear binding to de-energized cells; binding of tetraphenylphosphonium and tetraphenylarsonium was nonlinear but was not saturated at the highest concentration tested (1mm). The usual assumption, that association of the cation with cell surfaces is saturated and so can be estimated on de-energized cells, therefore leads to undercorrected estimates of cytoplasmic uptake inB. subtilis, and hence to overestimates of membrane potential. We describe a more realistic procedure, in which the estimate of extent of binding is based on a mean aqueous concentration related both to the external concentration and to the much higher internal concentration that exists in energized cells. Using this procedure we estimate the membrane potential inB. subtilis to be 120 mV, inside-negative. The procedure is of general applicability, and should yield more accurate estimates of membrane potential in any system where there is significant potential-dependent binding.Work performed while on sabbatical leave from Department of Biology, Ben-Gurion University of the Negev, Beer-Sheva, Israel.     

Interaction of phosphate with monovalent cation uptake in yeast.

The uptake of monovalent cations by yeast via the monovalent cation uptake mechanism is inhibited by phosphate. The inhibition of Rb+ uptake shows saturation kinetics and the phosphate concentration at which half-maximal inhibition is observed is equal to the Km of phosphate for the sodium-independent phosphate uptake mechanism. The kinetic coefficients of Rb+ and TI+ uptake are affected by phosphate: the maximal rate of uptake is decreased and the apparent affinity constants for the translocation sites are increased. In the case of Na+ uptake, the inhibition by phosphate may be partly or completely compensated by stimulation of Na+ uptake via a sodium-phosphate cotransport mechanism. Phosphate effects a transient stimulation of the efflux of the lipophilic cation dibenzyldimethylammonium from preloaded yeast cells and a transient inhibition of dibenzyldimethylammonium uptake. Possibly, the inhibition of monovalent cation uptake in yeast can be explained by a transient depolarization of the cell membrane by phosphate.     

A critical assessment of the use of lipophilic cations as membrane potential probes

A critical review has been made of the literature on the use of lipophilic cations, such as triphenylmethyl phosphonium (TPMP⁺) as membrane potential probes in prokaryotes, uekaryote organelles in vitro, and eukaryote cells. An ideal lipophilic cation should be capable of penetrating through a biological membrane and obey the Nernst equation between a membrane bound phase and its environment. Many different forms of the Nernst equation are presented, useful in the calculation equilibrium potentials of lipophilic cations across membranes. Lipophilic cations appear to behave as valid membrane potential probes in prokaryotes and eukaryote organelles in vitro and even in vivo although some technical difficulties may be involved. On the other hand in valid forms of the Nernst equation have often been used to calculate the equilibrium potential of lipophilic cations across the plasma membranes of eukaryotic cells. In particular, the problem of intracellular compartmentation of lipophilic cations has often not been appreciated. Lipophilic cations do not appear to behave as reliable plasma membrane potential probes in eukaryotic cells. Some other avenues are discussed which might be useful in the determination of the plasma membrane potentials of small eukaryotic cells, e.g. the use of lipophilic anions as membrane potential probes.     

Transmembrane distribution of lipophilic cations in response to an electrochemical potential in reconstituted cytochromec oxidase vesicles and in vesicles exhibiting a potassium ion diffusion potential

It has been shown previously that biogenic amines and a number of pharmaceutical agents can redistribute across vesicle membranes in response to imposed potassium ion or proton gradients. Surprisingly, drug accumulation is observed for vesicles exhibiting either a pH gradient (interior acidic) or a membrane potential (interior negative), implying that these compounds can traverse the lipid bilayer as either the neutral or charged species. This interpretation, however, is complicated by the fact that vesicles exhibiting a membrane potential (interior negative) accumulate protons in response to this potential, thereby creating a pH gradient (interior acidic). This raises the possibility that in both vesicle systems drug redistribution occurs in response to the proton gradient present. We have therefore compared the uptake of several lipophilic cations by reconstituted cytochromec oxidase vesicles and by similar vesicles exhibiting a potassium ion diffusion potential. While turnover of the oxidase generates a membrane potential of comparable magnitude to the potassium ion diffusion system, it is associated with a proton gradient of opposite polarity (interior basic). Both systems show rapid uptake of the permanently charged lipophilic cation, tetraphenylphosphonium, but only the potassium ion diffusion system accumulates the lipophilic amines doxorubicin and propranolol. This provides compelling evidence that such weak bases redistribute only in response to pH gradients and not membrane potential.     

Reduction in accumulation of [3H]triphenylmethylphosphonium cation in neuroblastoma cells caused by optical probes of membrane potential: Evidence for interactions between carbocyanine dyes and lipophilic anions

The accumulation of [³H]triphenylmethylphosphonium cation in neuroblastoma N1E 115 cells in the presence of tetraphenylboron is reduced by 3,3′-diethylthiadicarbocyanine iodide and by 3,3′-dipropylthiadicarbocyanine iodide. This reduction in uptake of the lipophilic cation is not due to the carbocyanine dyes depolarizing the plasma membrane of these cells but due to an interaction between the carbocyanine dyes and tetraphenylboron leaving less of the lipophilic anion free in solution to assist uptake of the lipophilic cation. This interaction is shown to have a 1:1 stoicheiometry.     

Interaction of phosphate with monovalent cation uptake in yeast

The uptake of monovalent cations by yeast via the monovalent cation uptake mechanism is inhibited by phosphate. The inhibition of Rb⁺ uptake shows saturation kinetics and the phosphate concentration at which halfmaximal inhibition is observed is equal to the K_m of phosphate for the sodiumindependent phosphate uptake mechanism. The kinetic coefficients of Rb⁺ and Tl⁺ uptake are affected by phosphate: the maximal rate of uptake is decreased and the apparent affinity constants for the translocation sites are increased.In the case of Na⁺ uptake, the inhibition by phosphate may be partly or completely compensated by stimulation of Na⁺ uptake via a sodium-phosphate cotransport mechanism.Phosphate effects a transient stimulation of the efflux of the lipophilic cation dibenzyldimenthylammonium from preloaded yeast cells and a transient inhibition of dibenzyldimethylammonium eptake. Possibly, the inhibition of monovalent cation uptake in yeast can be explained by a transient depolarization of the cell membrane by phosphate.     

Mitochondrial accumulation of a lipophilic cation conjugated to an ionisable group depends on membrane potential, pH gradient and pK a: implications for the design of mitochondrial probes and therapies

Mitochondria play key roles in a broad range of biomedical situations, consequently there is a need to direct bioactive compounds to mitochondria as both therapies and probes. A successful approach has been to target compounds to mitochondria by conjugation to lipophilic cations, such as triphenylphosphonium (TPP), which utilize the large mitochondrial membrane potential (Δψ_m, negative inside) to drive accumulation. This has proven effective both in vitro and in vivo for a range of bioactive compounds and probes. However so far only neutral appendages have been targeted to mitochondria in this way. Many bioactive functional moieties that we would like to send to mitochondria contain ionisable groups with pK _a in the range that creates an assortment of charged species under physiological conditions. To see if such ionisable compounds can also be taken up by mitochondria, we determined the general requirements for the accumulation within mitochondria of a TPP cation conjugated to a carboxylic acid or an amine. Both were taken up by energised mitochondria in response to the protonmotive force. A lipophilic TPP cation attached to a carboxylic acid was accumulated to a greater extent than a simple TPP cation due to the interaction of the weakly acidic group with the pH gradient (ΔpH). In contrast, a lipophilic TPP cation attached to an amine was accumulated less than the simple cation due to exclusion of the weakly basic group by the ΔpH. From these data we derived a simple equation that describes the uptake of lipophilic cations containing ionisable groups as a function of Δψ_m, ΔpH and pK _a. These findings may facilitate the rational design of additional mitochondrial targeted probes and therapies.     

Effect of membrane surface charge on nickel uptake by purified mung bean root protoplasts

The influence of membrane surface charge on cation uptake was investigated in protoplasts prepared from roots of mung bean (Vigna radiata L.). Confocal laser scanning microscopy showed that a fluorescent trivalent cation accumulated to very high concentrations at the surface of the protoplasts when they were incubated in medium containing low concentrations of Ca or other cations, but that this accumulation could be completely reversed by suppression of membrane surface negativity by high cation concentrations. Influx of 63Ni was strongly reduced by a range of divalent cations. Increasing the Ca concentration in the medium from 25 microM to 10 mM inhibited 63Ni influx by more than 85%. 63Ni influx was also inhibited by 85% by reducing the pH from 7 to 4. Computation of the activity of Ni at the membrane surface under the various treatment conditions showed that Ni uptake was closely correlated with its activity at the membrane surface but not with its concentration in the bulk medium. It was concluded that the effects on Ni uptake of addition of monovalent, divalent and trivalent cations, and of variations in pH are all consistent with the proposition that the activity of Ni at the membrane surface is the major determinant of the rate of Ni influx into mung bean protoplasts. It is proposed that the surface charge on the plasma membrane will influence the membrane transport of most charged molecules into cells.     

Dissociation of H + extrusion from K+ uptake by means of lipophilic cations

Abstract Dissociation of active H⁺ extrusion (?ΔH⁺) from K⁺ uptake in pea and maize root segments was attempted by substituting K⁺ in the incubation medium with lipophilic cations assumed to enter the cell by passive, non-specific, permeation through the lipid component of the plasmalemma. Among the compounds tested, tributylbenzylammonium significantly stimulated ?ΔH⁺ in the absence of other monovalent cations in the medium. This effect was much more evident when the experiment was carried out in the presence of fusicoccin, which strongly stimulates proton extrusion and monovalent cation uptake, and hyperpolarizes the trans-membrane electric potential in these materials. Also the lipophilic cations tetraphenylphosphonium, dimethyldibenzylammonium and hexylguanidine markedly stimulated FC-promoted ?ΔH⁺. Octylguanidine at a low concentration induced an early stimulation followed by a strong inhibition of ?ΔH⁺. A complete lack of additivity was observed between the effects of lipophilic cations and that of K⁺ on H⁺ extrusion. Lipophilic cations severely inhibited K⁺ uptake. These data are interpreted as supporting the view of an electric, rather than a chemical, (namely, involving the same carrier system) nature of the coupling of active H⁺ extrusion with K⁺ influx.     

The yeast SR protein kinase Sky1p modulates salt tolerance,membrane potential and the Trk1,2 potassium transporter

Protein kinases dedicated to the phosphorylation of SR proteins have been implicated in the processing and nuclear export of mRNAs. Here we demonstrate in Saccharomyces cerevisiae their participation in cation homeostasis. A null mutant of the single yeast SR protein kinase Sky1p is viable but exhibits increased tolerance to diverse toxic cations such as Na(+), Li(+), spermine, tetramethylammonium, hygromycin B and Mn(2+). This pleiotropic phenotype correlates with reduced accumulation of cations, suggesting a decrease in membrane electrical potential. Genetic analysis and Rb(+) uptake measurements indicate that Sky1p modulates Trk1,2, the high-affinity K(+) uptake system of yeast and a major determinant of membrane potential.     

Co-transport of anions and neutral solutes with cations across charged biological membranes. Effects of surrface potential on uptake kinetics

General rate equations have been developed for the co-transport of an anion with one or two cations across a negatively charged biological membrane. The effects of surface potential on the kinetical parameters of co-transport of monovalent anions with monovalent cations have been investigated in more detail. The influence of changes in the surface potential on ion uptake kinetics appears to be markedly affected by the properties of the co-transport system. This can be shown by investigating boundary cases of the general model, namely (a) random order of binding of the ions, (b) anion binds before cations, (c) cations bind before anion. Since the effects of the surface potential are different for these three cases, these effects might serve as (additional) discrimination criteria.The effect of the surface potential on anion uptake kinetics via a co-transport system to which two cations can bind is rather complex: maxima or minima of the apparent affinity constant K_m of anion uptake may occur. Not only the magnitude of the effect of changes in the surface potential, but also its direction (stimulation, inhibition), is influenced by the co-substrate (cation) concentration. Such effects may also occur if only one cation can bind to the translocator, provided that OH^? ions compete for the anion transport site.In addition, the case of co-transport of a neutral solute with a monovalent cation has been investigated. It has been shown, that monovalent cation has been investigated. It has been shown, that also in this case, the effect of changes in the surfaces potential is affected by the order of binding of the substrates to the translocator.     

Permeability of Lipophilic Cations

The permeability (P) of a lipophilic cation, triphenylmethylphosphonium(TPMP⁺) which is frequently used as a membrane potential probe,has been measured in Chara australis (Charophyceae). P_TPMP+across biological membranes is usually thought to be very highbut this is not the case across the plasmalemma of Chara. Thepermeability of TPMP⁺ across the plasmalemma was found to betypical of inorganic cations, about 1.0 nm s^–1. Estimateswere made of the permeability of lipophilic cations across someother cell membranes, based on previously published work. Thepermeability of TPMP⁺ across the plasma membranes of the redalga, Griffithsia monilis and the blue-green alga, Anabaenavariabilis was about 2–5 nm s^–1. The permeabilityof TPMP⁺ across the plasma membranes of eukaryotes and prokaryotesappears to be similar. The permeability of lipophilic cationsacross the cristae of isolated mitochondria are exceptionallyhigh, about 170 nm s^–1. TPMP⁺ did not behave as a thiamineanalogue in Chara, unlike in the case of yeast. The means ofentry of TPMP⁺ into the Chara cell, driven by the electrochemicalgradient across the plasmalemma, has not been identified. Thepresence of a second lipophilic cation probe, DDA⁺ (dibenzyldimethylammonium),caused a decrease in the uptake flux of TPMP⁺; this suggeststhat the two lipophilic cations compete for the same site atthe surface of the plasmalemma. Key words: Chara australis, TPMP⁺, Permeability, Lipophilic cation     

Kinetics of Ca2+ and Sr2+ uptake by yeast. Effects of pH, cations and phosphate.

The uptake of Ca2+ and Sr2+ by the yeast Saccharomyces cerevisiae is energy dependent, and shows a deviation from simple Michaelis-Menten kinetics. A model is discussed that takes into account the effect of the surface potential and the membrane potential on uptake kinetics. The rate of Ca2+ and Sr2+ uptake is influenced by the cell pH and by the medium pH. The inhibition of uptake at low concentration of Ca2+ and Sr2+ at low pH may be explained by a decrease of the surface potential. The inhibition of Ca2+ and Sr2+ uptake by monovalent cations is independent of the divalent cation concentration. The inhibition shows saturation kinetics, and the concentration of monovalent cation at which half-maximal inhibition is observed, is equal to the affinity constant of this ion for the monovalent cation transport system. The inhibition of divalent cation uptake by monovalent cations appears to be related to depolarization of the cell membrane. Phosphate exerts a dual effect on uptake of divalent cations: and initial inhibition and a secondary stimulation. The inhibition shows saturation kinetics, and the inhibition constant is equal to the affinity constant of phosphate for its transport mechanism. The secondary stimulation can only partly be explained by a decrease of the cell pH, suggesting interaction of intracellular phosphate, or a phosphorylated compound, with the translocation mechanism.     

A resonance Raman and electronic absorption probe of membrane energization. Quinaldine red in cells of Streptococcus faecalis.

Resonance Raman and electronic absorption spectra were used to show that the state of an amphiphilic cation, relative to dilute aqueous solution, changes when it is accumulated by cells of Streptococcus faecalis when they are energized. The general characteristics of the cation employed, quinaldine red, closely paralleled those of other amphiphilic cations which have been used to measure membrane potential. A major aspect of the change is that in sodium-loaded cells, essentially all of the quinaldine red accumulated as the result of energization forms a strong bond with an anionic group. This binding is similar to that which occurs for the basal level of quinaldine red taken up in nonenergized cells. Ionic binding was detected using resonance Raman spectroscopy through shifts associated with a N+ parallel C--C parallel C stretching vibration to lower frequency on uptake. Another aspect of the change in state is that the cell-localized probe cation can aggregate while ionically bonded in a card pack fashion, the transition dipoles being parallel. A combination of resonance Raman and electronic absorption spectroscopy was used to characterize this aggregation. The aggregates were estimated to contain at least five quinaldine red cations at or near van der Waals contact, and the presence of other molecules, such as phospholipids, could not be excluded. Aggregation effects are complex depending on the ratio of cells to probe cation, and on energization. The site of binding is suggested to be the lipid bilayer region of the plasma membrane on the basis of experiments with liposomes and other model systems. In addition, some quinaldine red may be present in the cytoplasm in an aggregated, ionically bound form. The change in state on uptake following energization seems to be associated with a membrane potential, similar spectral and uptake effects being produced by an artificially generated membrane potential in cells and liposomes. The results show that membrane potential cannot be computed in a simple manner from the distribution of quinaldine red between cells and medium, assuming that the thermodynamic activity coefficient of cell-localized material is identical with that in dilute aqueous solution. However, uptake as well as subsequent ionic binding of quinaldine red seems to be related to potential in an as yet undefined manner.     

Control of the Ca2+-triggered bioluminescence of Veretillum cynomorium lumisomes.

Calcium ions can trigger an emission of light from Veretillum cynomorium lumisomes (bioluminescent vesicles) under conditions where they are not lysed. This process does not require a metabolically-linked source of energy, but is dependent upon the nature of the ions present inside and outside the vesicles. The Ca2+-triggered bioluminescence is stimulated by an asymmetrical distribution of cations or anions. Either high internal sodium or high external chloride is required for the maximal effect. When sodium is present outside the structure and potassium inside, the slow inward diffusion of calcium is decreased. Unbalanced diffusion of internal cations also stimulates the bioluminescence, suggesting control of the calcium influx by an electrochemical gradient. It is assumed that rapid outward diffusion of sodium or inward diffusion of chloride generates an electrical potential difference (inside negative) which drives the Ca2+-influx. With purified lumisomes it has been shown that Ca2+-triggered bioluminescence and calcium uptake (presumably net uptake) were correlated. In two instances uptake of the lipophilic cation dibenzyldimethylammonium has given direct evidence for the existence of a potential difference. With NaCl-loaded vesicles, it has not been possible to demonstrate an uptake of lipophilic cations but experiments with 22Na and 42D indicated a higher rate of sodium efflux, in accord with the proposed hypothesis.     

A novel mechanism of ion homeostasis and salt tolerance in yeast: the Hal4 and Hal5 protein kinases modulate the Trk1-Trk2 potassium transporter

The regulation of intracellular ion concentrations is a fundamental property of living cells. Although many ion transporters have been identified, the systems that modulate their activity remain largely unknown. We have characterized two partially redundant genes from Saccharomyces cerevisiae, HAL4/SAT4 and HAL5, that encode homologous protein kinases implicated in the regulation of cation uptake. Overexpression of these genes increases the tolerance of yeast cells to sodium and lithium, whereas gene disruptions result in greater cation sensitivity. These phenotypic effects of the mutations correlate with changes in cation uptake and are dependent on a functional Trk1-Trk2 potassium transport system. In addition, hal4 hal5 and trk1 trk2 mutants exhibit similar phenotypes: (i) they are deficient in potassium uptake; (ii) their growth is sensitive to a variety of toxic cations, including lithium, sodium, calcium, tetramethylammonium, hygromycin B, and low pH; and (iii) they exhibit increased uptake of methylammonium, an indicator of membrane potential. These results suggest that the Hal4 and Hal5 protein kinases activate the Trk1-Trk2 potassium transporter, increasing the influx of potassium and decreasing the membrane potential. The resulting loss in electrical driving force reduces the uptake of toxic cations and improves salt tolerance. Our data support a role for regulation of membrane potential in adaptation to salt stress that is mediated by the Hal4 and Hal5 kinases.     

Evidence for a negative membrane potential and for movement of C1- against its electrochemical gradient in the ascomycete Neocosmospora vasinfecta.

The iodides of three lipid-soluble cations (dibenzyldimethylammonium; tribenzylmethylammonium, TBMA+; ethyldimethylbenzylammonium) were synthesized by the reaction of 14C-labeled methyl or 14C-labeled ethyl iodide with the appropriate secondary of tertiary amine and used in an attempt to measure the transmembrane electrical potential difference in Neocosmospora. Only mycelium containing high levels of Na+ accumulated measureable amounts of these cations and only above pH 6. Uptake was reduced in the presence of exogenous K+, Na+, Mg2+, or tris(hydroxymethyl)aminomethane. The velocity of TBMA+ uptake was proportional to its concentration between 46 and 427 muM. Neither the rate nor the extent of TBMB+ uptake was greatly affected by the presence of a fivefold excess of either dibenzyldimethylammonium or ethyldimethylbenzylammonium, even though these cations were themselves accumulated. The uncoupler m-chlorophenylhydrazone induced loss of previously accumulated TBMA+ from the mycelium. Anaerobiosis and cold (5 degrees C) temperature both inhibited TBMA+ uptake but did not induce the loss of previously accumulated TBMA+. The uptake of lipophilic cations by Na+-rich mycelium indicated a minimum transmembrane electrical potential of -60 to -70 mV (inside negative). Net uptake of these cations appeared to be strongly influenced by the availability of endogenous exchangeable cations and by the presence of other exogenous cations, as well as by the membrane potential. Despite these limitations, transport of C1- by Na+-rich mycelium appeared to take place against the electrochemical gradient for C1-.     

Characterization of tetraethylammonium uptake across the basolateral membrane of the Drosophila Malpighian (renal) tubule

Basolateral transport of the prototypical type I organic cation tetraethylammonium (TEA) by the Malpighian tubules of Drosophila melanogaster was studied using measurements of basolateral membrane potential (V(bl)) and uptake of [(14)C]-labeled TEA. TEA uptake was metabolically dependent and saturable (maximal rate of mediated TEA uptake by all potential transport processes, reflecting the total transport capacity of the membrane, 0.87 pmol.tubule(-1).min(-1); concentration of TEA at 0.5 of the maximal rate of TEA uptake value, 24 muM). TEA uptake in Malpighian tubules was inhibited by a number of type I (e.g., cimetidine, quinine, and TEA) and type II (e.g., verapamil) organic cations and was dependent on V(bl). TEA uptake was reduced in response to conditions that depolarized V(bl) (high-K(+) saline, Na(+)-free saline, NaCN) and increased in conditions that hyperpolarized V(bl) (low-K(+) saline). Addition of TEA to the saline bathing Malpighian tubules rapidly depolarized the V(bl), indicating that TEA uptake was electrogenic. Blockade of K(+) channels with Ba(2+) did not block effects of TEA on V(bl) or TEA uptake indicating that TEA uptake does not occur through K(+) channels. This is the first study to provide physiological evidence for an electrogenic carrier-mediated basolateral organic cation transport mechanism in insect Malpighian tubules. Our results also suggest that the mechanism of basolateral TEA uptake by Malpighian tubules is distinct from that found in vertebrate renal tubules.     

Tetraphenylphosphonium is an indicator of negative membrane potential in Candida albicans

The characteristics of the uptake of lipophilic cations tetraphenylphosphonium (TPP+) into Candida albicans have been investigated to establish whether TPP+ can be used as a membrane potential probe for this yeast. A membrane potential (delta psi, negative inside) across the plasma membrane of C. albicans was indicated by the intracellular accumulation of TPP+. The steady-state distribution of TPP+ was reached within 60 min and varied according to the expected changes of delta psi. Agents known to depolarize membrane potential caused a rapid and complete efflux of accumulated TPP+. The initial influx of TPP+ was linear over a wide range of TPP+ concentrations (2.5-600 microM), indicating a non mediated uptake. Thus, TPP+ is a suitable delta psi probe for this yeast.