Characterization of osmotically activated ion currents in the membrane of cytoplasmic droplets from Phycomyces blakesleeanus Burgeff sporangiophores.

Membrana citoplazmatskih kapi dobijenih iz rastućih sporangiofora gljive Phycomyces blakesleeanus Burgeff je jedan od samo dva za sada poznata model sistema u kojima se mogu ispitivati elektrofiziološka svojstva plazma membrane filamentoznih gljiva (drugi model sistem je sluzavi mutant gljive Neurospora crassa). Pokazano je da bar 20% citoplazmatskih kapi nakon 2 h formira ćelijski zid što ukazuje da njihova membrana odgovara plazma membrani hife. Eksperimentima prikazanim u disertaciji je po prvi put izvršeno registrovanje struja metodom nametnute voltaže u konfiguraciji cela ćelija na model sistem membrane citoplazmatskih kapi. Ovaj način snimanja jonskih struja daje uvid u tipove i osobine dominantnih ukupnih struja prisutnih u ispitivanoj membrani. Okarakterisan je odgovor membrane na hipoosmotske uslove sredine, izazvane dijalizom čitave kapi hiperosmotskim rastvorom. Za razliku od familije struja u izoosmotskim uslovima, kojima dominiraju pasivna svojstva membrane, male konduktivnosti, u hipoosmotskim uslovima je karakteristično dominantno prisustvo izlazno ispravljene brzoinaktivirajuće trenutne struje (IRIS), aktivirane na depolarišućim potencijalima. Pored IRIS, prisutne u svakoj ispitivanoj membrani citoplazmatskih kapi čija je maksimalna gustina struje na +70 mV iznosila 129 ± 14 pA/pF (n=30), u 73% registrovanih odgovora je bila primetna i sporoaktivirajuća ulazna struja, gustine 99 ± 11 pA/pF na -150 mV (n=22) koja nije dalje ispitivana. IRIS je prava osmotski aktivirana struja jer: 1. ima dozno zavisnu osetljivost na osmotski stimulus; 2. promena sredine iz hipoosmotskih u hiperosmotske trenutno gasi IRIS. Prema svojim osobinama IRIS se jasno razlikuje od svih do sada opisanih jonskih struja kod gljiva, ali pokazuje brojne sličnosti sa zapreminom regulisanom anjonskom strujom (VRAC- volume regulated anionic channel) kod kičmenjaka. Ove sličnosti su sledeće: 1. aktivacija u uslovima osmotski izazvanog povećanja zapremine; 2. umereno izlazno ispravljanje, sa naelektrisanjem vratnica od zg = 0,82 ± 0,1; 3. voltažno i vremenski zavisna inaktivacija na pozitivnim potencijalima i oporavak od inaktivacije na negativnim potencijalima; 4. izražena selektivnost za anjone u odnosu na katjone sa karakterističnom sekvencom provodljivosti koja odgovara Ajsmanovoj seriji I (I-: Cl- : HCO3-: glukonat- : glutamat- = 1,4: 1: 0,25: 0,01: 0,088); 5. progresivno smanjenje amplitude struje u vremenu, koje usporava unutarćelijski ATP; 6. aktivacija nehidrolizujućim analogom GTP u izoosmotskim uslovima, kao i kod VRAC, ukazujući da se IRIS aktivira putem nekog GTP-zavisnog signalnog puta. 7. smanjenje struje u prisustvu jona magnezijuma sa unutarćelijske strane...Cytoplasmic droplet membrane obtained from growing sporangiophore of fungus Phycomyces blakesleeanus Burgeff is one out of two known exerimental models for electrophysiological exploration of plasma membrane properties of filamentous fungi (the other being Neurospora crassa slime mutant). At least 20 % of cytoplasmic droplets forms cell wall after 2 h, confirming that their membrane coresponds functionally to hyphal plasma membrane. This is the first patch-clamp registration in whole cell configuration obtained on cytoplasmic droplet membrane model sistem. This configuration gives insight into types and properties of dominant ion currents present on membrane under investigation. The membrane response to hypoosmotic stimuli, performed by whole droplet dialysis by hyperosmotic solution, is characterised in detail. In contrast to familly of currents under isoosmotic conditions, that are dominated by passive membrane properties and small conductances, under hypoosmotic conditions there is prominent outwardly rectified fast-inactivating instantenous current (ORIC) that is activated at depolarisied potentials. In addition to ORIC that was present in every doplet examined (with average maximal current density at +70 mV = 129 ± 14 pA/pF (n=30)), in 73% of obtained responses there was noticable an additional current, slowly activating inward current, current density at -150 mV = 99 ± 11 pA/pF (n=22). The inward current was not examined in more detail. IRIS is true osmoticaly activated current according to following properties: 1. Its osmotic sensitivity is dose-dependent; 2. It shuts down immidiately upon change from hypoosmotic to hyperosmotic enviroment. IRIS is clearly different from other known fungal ion channels, but shares a number of features with vertebrate VRAC (volume regulated anionic channel): 1. activation under conditions of osmotially induced volume increase; 2. moderate outward rectification with gate charge zg = 0.82 ± 0.1; 3. time- and voltage-dependent inactivation at positive potentials and recovery from inactivation at negative potentials; 4. distinct selectivity for anions over cations, with characteristic permeability sequence corresponding to Aismann series I (I-: Cl- : HCO3-: glukonat- : glutamat- = 1.4: 1: 0.25: 0.01: 0.088); 5. time dependent current shut down that is slowed down by intracellular ATP; 6. activation by unhydrolizable GTP analogs under isoosmotic conditions, pointing to ORIC being activated through GTP-dependent cascade; 7. decrease of current in the presence of intracellular magnesium ions..

Characterization of osmotically activated ion currents in the membrane of cytoplasmic droplets from Phycomyces blakesleeanus Burgeff sporangiophores.

Membrana citoplazmatskih kapi dobijenih iz rastućih sporangiofora gljive Phycomyces blakesleeanus Burgeff je jedan od samo dva za sada poznata model sistema u kojima se mogu ispitivati elektrofiziološka svojstva plazma membrane filamentoznih gljiva (drugi model sistem je sluzavi mutant gljive Neurospora crassa). Pokazano je da bar 20% citoplazmatskih kapi nakon 2 h formira ćelijski zid što ukazuje da njihova membrana odgovara plazma membrani hife. Eksperimentima prikazanim u disertaciji je po prvi put izvršeno registrovanje struja metodom nametnute voltaže u konfiguraciji cela ćelija na model sistem membrane citoplazmatskih kapi. Ovaj način snimanja jonskih struja daje uvid u tipove i osobine dominantnih ukupnih struja prisutnih u ispitivanoj membrani. Okarakterisan je odgovor membrane na hipoosmotske uslove sredine, izazvane dijalizom čitave kapi hiperosmotskim rastvorom. Za razliku od familije struja u izoosmotskim uslovima, kojima dominiraju pasivna svojstva membrane, male konduktivnosti, u hipoosmotskim uslovima je karakteristično dominantno prisustvo izlazno ispravljene brzoinaktivirajuće trenutne struje (IRIS), aktivirane na depolarišućim potencijalima. Pored IRIS, prisutne u svakoj ispitivanoj membrani citoplazmatskih kapi čija je maksimalna gustina struje na +70 mV iznosila 129 ± 14 pA/pF (n=30), u 73% registrovanih odgovora je bila primetna i sporoaktivirajuća ulazna struja, gustine 99 ± 11 pA/pF na -150 mV (n=22) koja nije dalje ispitivana. IRIS je prava osmotski aktivirana struja jer: 1. ima dozno zavisnu osetljivost na osmotski stimulus; 2. promena sredine iz hipoosmotskih u hiperosmotske trenutno gasi IRIS. Prema svojim osobinama IRIS se jasno razlikuje od svih do sada opisanih jonskih struja kod gljiva, ali pokazuje brojne sličnosti sa zapreminom regulisanom anjonskom strujom (VRAC- volume regulated anionic channel) kod kičmenjaka. Ove sličnosti su sledeće: 1. aktivacija u uslovima osmotski izazvanog povećanja zapremine; 2. umereno izlazno ispravljanje, sa naelektrisanjem vratnica od zg = 0,82 ± 0,1; 3. voltažno i vremenski zavisna inaktivacija na pozitivnim potencijalima i oporavak od inaktivacije na negativnim potencijalima; 4. izražena selektivnost za anjone u odnosu na katjone sa karakterističnom sekvencom provodljivosti koja odgovara Ajsmanovoj seriji I (I-: Cl- : HCO3-: glukonat- : glutamat- = 1,4: 1: 0,25: 0,01: 0,088); 5. progresivno smanjenje amplitude struje u vremenu, koje usporava unutarćelijski ATP; 6. aktivacija nehidrolizujućim analogom GTP u izoosmotskim uslovima, kao i kod VRAC, ukazujući da se IRIS aktivira putem nekog GTP-zavisnog signalnog puta. 7. smanjenje struje u prisustvu jona magnezijuma sa unutarćelijske strane...Cytoplasmic droplet membrane obtained from growing sporangiophore of fungus Phycomyces blakesleeanus Burgeff is one out of two known exerimental models for electrophysiological exploration of plasma membrane properties of filamentous fungi (the other being Neurospora crassa slime mutant). At least 20 % of cytoplasmic droplets forms cell wall after 2 h, confirming that their membrane coresponds functionally to hyphal plasma membrane. This is the first patch-clamp registration in whole cell configuration obtained on cytoplasmic droplet membrane model sistem. This configuration gives insight into types and properties of dominant ion currents present on membrane under investigation. The membrane response to hypoosmotic stimuli, performed by whole droplet dialysis by hyperosmotic solution, is characterised in detail. In contrast to familly of currents under isoosmotic conditions, that are dominated by passive membrane properties and small conductances, under hypoosmotic conditions there is prominent outwardly rectified fast-inactivating instantenous current (ORIC) that is activated at depolarisied potentials. In addition to ORIC that was present in every doplet examined (with average maximal current density at +70 mV = 129 ± 14 pA/pF (n=30)), in 73% of obtained responses there was noticable an additional current, slowly activating inward current, current density at -150 mV = 99 ± 11 pA/pF (n=22). The inward current was not examined in more detail. IRIS is true osmoticaly activated current according to following properties: 1. Its osmotic sensitivity is dose-dependent; 2. It shuts down immidiately upon change from hypoosmotic to hyperosmotic enviroment. IRIS is clearly different from other known fungal ion channels, but shares a number of features with vertebrate VRAC (volume regulated anionic channel): 1. activation under conditions of osmotially induced volume increase; 2. moderate outward rectification with gate charge zg = 0.82 ± 0.1; 3. time- and voltage-dependent inactivation at positive potentials and recovery from inactivation at negative potentials; 4. distinct selectivity for anions over cations, with characteristic permeability sequence corresponding to Aismann series I (I-: Cl- : HCO3-: glukonat- : glutamat- = 1.4: 1: 0.25: 0.01: 0.088); 5. time dependent current shut down that is slowed down by intracellular ATP; 6. activation by unhydrolizable GTP analogs under isoosmotic conditions, pointing to ORIC being activated through GTP-dependent cascade; 7. decrease of current in the presence of intracellular magnesium ions..

Osmotically Activated Anion Current of Phycomyces Blakesleeanus—Filamentous Fungi Counterpart to Vertebrate Volume Regulated Anion Current

Studies of ion currents in filamentous fungi are a prerequisite for forming a complete understanding of their physiology. Cytoplasmic droplets (CDs), obtained from sporangiophores of Phycomyces blakesleeanus, are a model system that enables the characterization of ion currents in the native membrane, including the currents mediated by the channels not yet molecularly identified. Osmotically activated anionic current with outward rectification (ORIC) is a dominant current in the membrane of cytoplasmic droplets under the conditions of hypoosmotic stimulation. We have previously reported remarkable functional similarities of ORIC with the vertebrate volume regulated anionic current (VRAC), such as dose-dependent activation by osmotic difference, ion selectivity sequence, and time and voltage dependent profile of the current. Using the patch clamp method on the CD membrane, we further resolve VRAC-like ORIC characteristics in this paper. We examine the inhibition by extracellular ATP and carbenoxolone, the permeation of glutamate in presence of chloride, selectivity for nitrates, and activation by GTP, and we show its single channel behavior in excised membrane. We propose that ORIC is a functional counterpart of vertebrate VRAC in filamentous fungi, possibly with a similar essential role in anion efflux during cell volume regulation

ATP modulation of osmotically activated anionic current in the membrane of Phycomyces blakesleeanus sporangiophore

Abstract Ion channels are vital components of filamentous fungi signaling in communication with their environment. We exploited the ability of the apical region of growing sporangiophores of Phycomyces blakesleeanus to form membrane-enveloped cytoplasmic droplets (CDs), to examine ion currents in the filamentous fungi native plasma membrane. In hypoosmotic conditions, the dominant current in the CDs is ORIC, an osmotically activated, anionic, outwardly rectified, fast inactivating instantaneous current that we have previously characterized. Here, we examined the effect of ATP on ORIC. We show that CDs contain active mitochondria, and that respiration inhibition by azide accelerates ORIC inactivation. ATP, added intracellularly, reduced ORIC run-down and shifted the voltage dependence of inactivation toward depolarized potentials, in a manner that did not require hydrolysis. Notably, ATP led to slowing down of ORIC inactivation, as evidenced by an increased time constant of inactivation, τin, and slower decline of τin during prolonged recordings. Flavonoids (genistein and quercetin) had the effect on ORIC opposite to ATP, acting as current inhibitors, possibly by disrupting the stabilizing effect of ATP on ORIC. The integration of osmotic sensing with ATP dependence of the anionic current, typical of vertebrate cells, is described here for the first time in filamentous fungi

Vanadate influence on metabolism of sugar phosphates in fungus Phycomyces blakesleeanus.

The biological and chemical basis of vanadium action in fungi is relatively poorly understood. In the present study, we investigate the influence of vanadate (V5+) on phosphate metabolism of Phycomyces blakesleeanus. Addition of V5+ caused increase of sugar phosphates signal intensities in 31P NMR spectra in vivo. HPLC analysis of mycelial phosphate extracts demonstrated increased concentrations of glucose 6 phosphate, fructose 6 phosphate, fructose 1, 6 phosphate and glucose 1 phosphate after V5+ treatment. Influence of V5+ on the levels of fructose 2, 6 phosphate, glucosamine 6 phosphate and glucose 1, 6 phosphate (HPLC), and polyphosphates, UDPG and ATP (31P NMR) was also established. Increase of sugar phosphates content was not observed after addition of vanadyl (V4+), indicating that only vanadate influences its metabolism. Obtained results from in vivo experiments indicate catalytic/inhibitory vanadate action on enzymes involved in reactions of glycolysis and glycogenesis i.e., phosphoglucomutase, phosphofructokinase and glycogen phosphorylase in filamentous fungi

Growth inhibition of fungus Phycomyces blakesleeanus by anion channel inhibitors anthracene-9-carboxylic and niflumic acid attained through decrease in cellular respiration and energy metabolites

Increasing resistance of fungal strains to known fungicides has prompted identification of new candidates for fungicides among substances previously used for other purposes. We have tested the effects of known anion channel inhibitors anthracene-9-carboxylic acid (A9C) and niflumic acid (NFA) on growth, energy metabolism and anionic current of mycelium of fungus Phycomyces blakesleeanus. Both inhibitors significantly decreased growth and respiration of mycelium, but complete inhibition was only achieved by 100 and 500 μM NFA for growth and respiration, respectively. A9C had no effect on respiration of human NCI-H460 cell line and very little effect on cucumber root sprout clippings, which nominates this inhibitor for further investigation as a potential new fungicide. Effects of A9C and NFA on respiration of isolated mitochondria of P. blakesleeanus were significantly smaller, which indicates that their inhibitory effect on respiration of mycelium is indirect. NMR spectroscopy showed that both A9C and NFA decrease the levels of ATP and polyphosphates in the mycelium of P. blakesleeanus, but only A9C caused intracellular acidification. Outwardly rectifying, fast inactivating instantaneous anionic current (ORIC) was also reduced to 33±5 and 21±3% of its pre-treatment size by A9C and NFA, respectively, but only in the absence of ATP. It can be assumed from our results that the regulation of ORIC is tightly linked to cellular energy metabolism in P. blakesleeanus, and the decrease in ATP and polyphosphate levels could be a direct cause of growth inhibition.Microbiology (2017), 163(3): 364-37

Sugar phopsphates content in perchloric acid extracts of <i>P. blakesleeanus</i> mycelium.

a<p>compound proposed according to retention time and ³¹P NMR chemical shift.</p>b<p>sum of F6P, F1,6P and F2,6P.</p>c<p>relative increase of peak area normalized to the value in control.</p><p>*normalized to MDP signal area.</p

Influence of vanadium on SP content in perchloric acid extract of <i>P. blakeskeeanus</i> mycelia.

<p><b>A.</b> HPLC chromatograms obtained from control and V⁵⁺ treated mycelia. <b>B.</b> Changes in content of SP caused by V⁵⁺, and V⁴⁺, and control. Statistically significant differences are marked with asterix (n = 7, <i>P</i><0.05).</p

³¹P NMR spectra of <i>P. blakeskeeanus</i> mycelium after addition of: A. 24

<p> µ<b>mol/g_FW of G6P, F6P and G1P into mycelium treated with 80</b> µ<b>mol/g_FW V⁵⁺; B. 80</b> µ<b>mol/g_FW cAMP, 80</b> µ<b>mol/g_FW V⁵⁺, and 80</b> µ<b>mol/g_FW cAMP+80 </b><b>µmol/g_FW V⁵⁺.</b> Positions of added compounds are labeled with arrows.</p