H+ H2O ADP+Pi reducing equivalents ATP H+ Ions, metabolites H+ 1) The membrane-located ATPase systems of mitochondria and chloroplasts are hydro-dehydration systems with terminal specificities for water and ATP; and their normal function is to couple reversibly the translocation of protons across the membrane to the flow of anhydro-bond equivalents between water and the couple ATP/(ADP + Pi). 2) The membrane-located oxido-reduction chain systems of mitochondria and chloroplasts catalyse the flow of reducing equivalents, such as hydrogen groups and electron pairs, between substrates of different oxido-reduction potential; and their normal function is to couple reversibly the translocation of protons across the membrane to the flow of reducing equivalents during oxido-reduction. 3) There are present in the membrane of mitochondria and chloroplasts substrate-specific exchange- diffusion carrier systems that permit the effective reversible transmembrane exchange of anions against OH- and of cations against H+; and the normal normal function of these systems is to regulate the pH and osmotic differential across the membrane, and to permit entry and exit of essential metabolites (e.g., substrates and phosphate acceptor) without collapse of the membrane potential. 4) The systems of postulates 1, 2, and 3 are located in a specialized coupling membrane which has a low permeability to protons and to anions and cations generally. MITCHELL, P. Coupling of phosphorylation to electron and hydrogen transfer by a chemiosmotic type of mechanism. Nature 191:144–148, 1961. Nobel Prize in Chemistry 1978 reducing equivalents H2O H+ H+ ADP+Pi ATP Ions, metabolites H+

(Dy=yin-yout) Proton motive force (electrochemical gradient): ~200-220mV in isolated mitochondria (DY ~ 180mV) Electrochemical gradient for K+: for equilibrium [K+]o=150mM -> [K+]i=150M ??? Mitchell’s 4th postulate !!!!

Nigericin: H+/K+ exchanger FCCP: protonophor (~DNP) Valinomycin: K+ ionophor cholin-acetate K-acetate Na-acetate

Monovalens kation/H+ cserélők Na+ (Li+) szelektív cserélő Amilorid analógok (-) pH: széles optimum Mg2+: nem gátol Feladata: részt vesz a Na+-Ca2+ antiporterrel a Ca2+ effluxban H+-K+ antiporter (nem szelektív Na+,K+,Li+) pH: növekvő pH aktivál Mg2+: gátol Ozmotikus duzzadás aktivál

Monovales kation csatornák Na+-csatorna Ruthenium red, Mg2+, gilbenclamide (-) Nem-szelektív K+ csatorna Szelektív, ATP-szenzitív K+ csatorna ATP, Mg2+ jelenlétében (-) Diazoxide, krómkalim (+) Gilbenclamide, 5-hydroxydecanoate (-) S: bizonytalan in vivo szerep (Mg2+ gátlás) KATP: kardioprotektív hatás vs. Létezésének megkérdőjelezése K+ ciklus: a mitokondriális térfogat finom szabályzására adhat lehetőséget -> kapcsolat a metabolikus szabályozással.

FIG. 8. Pathways for Ca21 transport in energized mitochondria. ;, Redox-linked H1 pumps; UN, Ca21 uniporter; NICE, Na1-independent pathway for Ca21 efflux; NCE, Na1-dependent pathway for Ca21 efflux; NHE, H1/Na1 exchanger; RaM, rapid uptake mode; [Ca21]c, cytosolic Ca21 concentration; [Ca21]m, mitochondrial Ca21 concentration. For explanation, see text.

Ca2+ uniporter Rapid uptake mode Ca2+ elektroforetikus felvétele (szállított töltés: 2) Feszültségfüggés, Ca2+ aktiváció Km~10mM Ruthenium red, Ru360 (-) Kompetetív gátlók a csatornába kötődnek, transzportálódnak is: Sr2+,Mn2+,Ba2+ Allosztérikus gátlók: Mg2+,Mn2+, Spermin (+) ATP>ADP>AMP (-) Ismeretlen molekuláris identitás Rapid uptake mode Ca2+ felvétel [Ca2+]o~400nM körül Tranziens felvétel, Reset Ruthenium red, Ru360 (-, de csak nagyobb koncentrációban) ATP aktivál Mg2+ nem gátol Ismeretlen molekuláris identitás FIG. 8. Pathways for Ca21 transport in energized mitochondria. ;, Redox-linked H1 pumps; UN, Ca21 uniporter; NICE, Na1-independent pathway for Ca21 efflux; NCE, Na1-dependent pathway for Ca21 efflux; NHE, H1/Na1 exchanger; RaM, rapid uptake mode; [Ca21]c, cytosolic Ca21 concentration; [Ca21]m, mitochondrial Ca21 concentration. For explanation, see text.

Na+-független Ca2+-efflux (NICE) nH+-Ca2+ antiporter, n>2 vagy kapcsolt az elektron transzport lánchoz DYm szükséges Ruthenium Red inszenzitív Lassú és hamar telítődik Na+-függő Ca2+-efflux (NCE) 3Na+-Ca2+ antiporter DYm szükséges Ruthenium Red (-) Mg2+,Sr2+,Ba2+,Mn2+ (-) Amilorid, trifluoperazin, diltiazem, CGP-37157 (-) Mátrix pH függő (opt.= pH 7.6) Ca2+-cycling A Ca2+ efflux alacsony vmax-a és korai telítődése ellene hat A [Ca2+]matrix finom szabályzását teszi lehetővé (pl.: cardiomyocyta) Térbeli szegregáció ??? FIG. 8. Pathways for Ca21 transport in energized mitochondria. ;, Redox-linked H1 pumps; UN, Ca21 uniporter; NICE, Na1-independent pathway for Ca21 efflux; NCE, Na1-dependent pathway for Ca21 efflux; NHE, H1/Na1 exchanger; RaM, rapid uptake mode; [Ca21]c, cytosolic Ca21 concentration; [Ca21]m, mitochondrial Ca21 concentration. For explanation, see text.

10 mm Mitokondriális Ca2+-szenzitív fluoreszcens festék (x-rhod-1) stimulus: ATP (100mM, 6s) This is a raw fluorescenct image of X-rhod-1 loaded cells. Using the purinergic agonist ATP we evoked Ca2+ rise from the endoplasmic reticulum. ***click The red ATP sign shows the stimulation. As we see the mitochondrial network brightens up from the dark background, as they took up calcium. On can see, that the mitochondrial calcium dye x-rhod-1 is not specific for the mitochondria, it is present throughout the cytoplasm and the nucleus. As you see mitochondria took up calcium. … The Idea of highpass filtering technique was to find fine image details which are the mitochondria on the images. It was done by designing an apropriate highpass filter function, using filtering in Fourier domain. 10 mm

Mitokondriális kalciumfelvétel  lassabb kinetika ATP: purinerg agonista Gq->PLC->IP3  DYm függő FCCP: szétkapcsolószer  Na+/Ca2+-csere szerepe Just in brief, how did we validate the highpass filtering technique. The detailed description of the technique is just coming out of the press …cell calcium… Up here there is a typical Ca transient evoked by the ATP. The red shows mitochondrial calcium, the black the cytosolic . The plot shows relative fluorescence change normalized to 1 as a function of time in seconds. The clearest features of the mitochondrial signal are the delayed peak and slower decay fase. We blocked mitochondrial Ca2+ uptake by discharging of mito membrane potential by the uncoupler FCCP. The lack of mitochondrial Ca2+ elevation indicates selectivity. We also tested the effect of CGP37157 a blocker of MNCX, which caused a prolonged elevation of mitochondrial signal. The main adventage of highpass filtering over other techniques of mitochondrial calcium measurement is that the intensive fluorescent light enabled high spatial and temporal resolution acquisition. CGP-37157: Na-Ca cserélő gátlószere Cell Calcium 2001; 30.5: 311-321

Citrátkör aktiváció [Ca2+]matrix + Pyruvate dehydrogenase complex a-ketoglutarate Isocytrate +

Group I (modulator) (anti) Bcl-2, Bcl-xL, Bcl-w, Mcl-1, 1/Bfl1,Boo/Diva and Nrf3 BH1–BH4 domains C-terminal hydrophobic to attach ER & mitochondria Group II (output) (pro) Bax, Bak and Bok/Mtd No BH4 domain membrane permeabilization: pore formation (Bax tetramer + cardiolipin) Group III (sensor) (pro) Bid, Bad, Bik, Bim, Blk, Bmf, Hrk, Bnip3, Nix, Noxa and Puma responds to a wide variety of pro-apoptotic stimuli removal oftrophic factors cytoskeletal alterations DNA damage TRENDS in Cell Biology Vol.11 No.12 December 2001