Dominantne struje ćelijske membrane hifa i membrane citoplazmatskih kapi poreklom iz sporangiofora gljive Phycomyces blakesleeanus: karakterizacija izlazno-ispravljačke inaktivirajuće struje citoplazmatskih kapi

Abstract

Jonski kanali končastih gljiva su oblast istraživanja koja je jako slabo izučena, iako od ključne važnosti za razumevanje njihove fiziologije. Citoplazmatske kapi (CK) sporangiofora gljive Phycomyces blakesleeanus prethodno su uvedene kao model sistem za ispitivanje jonskih struja ćelijske membrane sporangiofora. Dominantna struja citoplazmatskih kapi u hipoosmotskim uslovima je izlazno-ispravljačka (rektifikujuća) inaktvirajuća struja – IRIS, koja nalikuje zapreminski regulisanoj anjonskoj struji kičmenjaka po biofizičkim osobinama među kojima je sekvenca selektivnosti, izlazno ispravljanje i specifičan obrazac inaktivacije na depolarišućim potencijalima. Drugi novorazvijeni model sistem za izučavanje jonskih kanala končastih gljiva su protoplasti dobijeni preciznim uklanjanjem ćelijskog zida hifa Phycomyces blakesleeanus femtosekundnim laserskim sistemom. U okviru ove disertacije okarakterisan je modulatorni efekat ATP-a na IRIS i prvi put su registrovane aktivnosti pojedinačnih kanala u osnovi IRIS u konfiguraciji „spolja spolja“. Pokazano je da CK produkuju ATP i funkcionalno odgovaraju plazmamembrani sporangiofora. ATP stabiliše otvoreno stanje kanala IRIS alosteričkom modulacijom, a najverovatnije ne utiče na aktivaciju IRIS, već samo umanjuje nivo inaktivacije. U prisustvu ATP IRIS se oporavlja od depolarizacijom-indukovane inaktivacije na naponski i vremenski zavisan način. Ispitan je farmakološki profil IRIS sa akcentom na molekulske vrste koje deluju na zapreminski-regulisan kanal kičmenjaka: flavonoide, vanćelijski ATP i karbenoksolon. Od jona sa fiziološkim značajem za gljivu, IRIS provodi nitrate, dok nije propustljiva za fosfate. Registrovan je obrazac inaktivacije IRIS na nivou pojedinačnih kanala, što je potvrđeno blokom vanćelijskim ATP-om i registracijom obrasca oporavka od inaktivacije. Provodljivost kanala u osnovi IRIS je 14 pS na 50-90 mV i primećeno je blago izlazno ispravljanje na nivou pore. Ispitana je efikasnost ostvarivanja prisnog kontakta mikropipete sa membranom protoplasta dobijenih ćelijskom hirurgijom i upoređena sa efikasnošću registracije sa citoplazmatskih kapi. Napravljen je preliminarni pregled aktivnosti jonskih struja u membrani protoplasta dobijenih hirurgijom. Dominanna jonska struja registrovana sa cele membrane protoplasta je ulazna inaktivirajuća struja, koja je najverovatnije nošena anjonima. Na nivou pojedinačnih kanala registrovane su aktivnosti kanala različitih provodljivosti, selektivnih za hlor, glutamat, više anjona, kalijum i kalcijum, sa dominatno prisutnim anjonskim strujama. Rezultati postavljaju osnov za ispitivanje sličnosti registrovanih struja sa već opisanim jonskim strujama u drugim gljivama i pronalaženje novih kanala specifičnih za gljive iz grupe kojoj P. blakesleeanus pripada.

Ion channels of filamentous fungi are an area of research that is very poorly studied, although it is of key importance for understanding their physiology. Cytoplasmic droplets derived from the sporangiophore of the fungus Phycomyces blakesleeanus were previously introduced as a model system for investigating ionic currents of the sporangiophore cell membrane. The dominant current recorded from cytoplasmic droplets in hypoosmotic conditions is the outwardly-rectifying inactivating current - IRIS, which resembles the volume-regulated anionic current of vertebrates in several biophysical properties, among which are the sequence of selectivity, outward rectification and a specific pattern of inactivation at depolarizing potentials. Another newly developed model system for studying ion channels of filamentous fungi are protoplasts obtained by precise removal of the hyphal cell wall of Phycomyces blakesleeanus by femtosecond laser system. Within this disertation, the modulatory effect of ATP on IRIS was characterized and for the first time the single channel activities of IRIS in the "outside-out" configuration were registered. It was shown that CKs produce ATP and functionally correspond to the plasma membrane of the sporangiophore. ATP stabilizes the open state of the IRIS channel by allosteric modulation, and most likely does not affect the activation of IRIS, but only reduces the level of inactivation. In the presence of ATP, IRIS recovers from depolarization-induced inactivation in a voltage and time-dependent manner. The pharmacological profile of IRIS was examined with an emphasis on molecular species that act on the volume-regulated channel of vertebrates: flavonoids, extracellular ATP and carbenoxolone. Of the ions with physiological importance for the fungus, IRIS conducts nitrates, while it is not permeable to phosphates. The pattern of IRIS inactivation at the level of individual channels was registered, which was confirmed by the block with extracellular ATP and by recording the pattern of recovery from inactivation. The conductance of IRIS at the single channel level is 14 pS at 50-90 mV and a slight outward rectification is observed at the pore level. The efficiency of making a tight seal of the micropipette with the membrane of protoplasts obtained by cell surgery was tested and compared with the efficiency of registration from cytoplasmic droplets. A preliminary review of the activity of ion currents in the membrane of protoplasts obtained by cell surgery was made. The dominant ionic current registered from the entire protoplast membrane is the inward inactivating current, which is most likely carried by anions. At the level of individual channels, the activities of channels of different conductances, selective for chlorine, glutamate, several anions, potassium and calcium, with dominantly present anion currents, were registered. The results set the basis for examining the similarity of registered currents with already described ion currents in other fungi and finding new channels specific to fungi from the group to which P. blakesleeanus belongs.