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

dc.contributor.advisorŽivić, Miroslav
dc.contributor.otherTodorović, Nataša
dc.contributor.otherStanić, Marina
dc.contributor.otherNikolić, Ljiljana
dc.contributor.otherCvetić-Antić, Tijana
dc.creatorKrižak, Strahinja V.
dc.date.accessioned2019-02-26T11:22:25Z
dc.date.available2019-02-26T11:22:25Z
dc.date.available2020-07-03T08:14:28Z
dc.date.issued2018-09-26
dc.identifier.urihttps://nardus.mpn.gov.rs/handle/123456789/10768
dc.identifier.urihttp://eteze.bg.ac.rs/application/showtheses?thesesId=6613
dc.identifier.urihttps://fedorabg.bg.ac.rs/fedora/get/o:19413/bdef:Content/download
dc.identifier.urihttp://vbs.rs/scripts/cobiss?command=DISPLAY&base=70036&RID=50761231
dc.description.abstractMembrana 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...sr
dc.description.abstractCytoplasmic 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...en
dc.formatapplication/pdf
dc.languagesr
dc.publisherУниверзитет у Београду, Биолошки факултетsr
dc.relationinfo:eu-repo/grantAgreement/MESTD/Basic Research (BR or ON)/173040/RS//
dc.rightsopenAccessen
dc.rights.urihttps://creativecommons.org/licenses/by-nc-nd/4.0/
dc.sourceУниверзитет у Београдуsr
dc.subjectPhycomyces blakesleaanussr
dc.subjectPhycomyces blakesleaanusen
dc.subjectelectrophysiologyen
dc.subjectanionic currenten
dc.subjecthypoosmotic conditionen
dc.subjectgrowthen
dc.subjectrespirationen
dc.subjectelektrofiziologijasr
dc.subjectanjonska strujasr
dc.subjecthipoosmotska sredinasr
dc.subjectrastsr
dc.subjectdisanjesr
dc.titleKarakterizacija osmotski aktiviranih jonskih struja u membrani citoplazmatskih kapi izolovanih iz sporangiofora gljive Phycomyces blakesleeanus Burgeffsr
dc.title.alternativeCharacterization of osmotically activated ion currents in the membrane of cytoplasmic droplets from Phycomyces blakesleeanus Burgeff sporangiophores.en
dc.typedoctoralThesisen
dc.rights.licenseBY-NC-ND
dc.identifier.fulltexthttps://nardus.mpn.gov.rs/bitstream/id/3339/Disertacija.pdf
dc.identifier.fulltexthttp://nardus.mpn.gov.rs/bitstream/id/3340/IzvestajKomisije19090.pdf
dc.identifier.fulltexthttps://nardus.mpn.gov.rs/bitstream/id/3340/IzvestajKomisije19090.pdf
dc.identifier.fulltexthttp://nardus.mpn.gov.rs/bitstream/id/3339/Disertacija.pdf
dc.identifier.rcubhttps://hdl.handle.net/21.15107/rcub_nardus_10768


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