Factors and processes involved in membrane potential build-up in yeast: diS-C3(3) assay.
No methods are currently available for fully reliable monitoring of membrane potential changes in suspensions of walled cells such as yeast. Our method using the Nernstian cyanine probe diS-C3(3) monitors even relatively fast changes in membrane potential delta psi by recording the shifts of probe fluorescence maximum lambda max consequent on delta psi-dependent probe uptake into, or exit from, the cells. Both increased [K+]out and decreased pHout, but not external NaCl or choline chloride depolarise the membrane. The major ion species contributing to the diS-C3(3)-reported membrane potential in S. cerevisiae are thus K+ and H+, whereas Na+ and Cl- do not perceptibly contribute to measured delta psi. The strongly pHout-dependent depolarisation caused by the protonophores CCCP and FCCP, lack of effect of the respiratory chain inhibitors rotenone and HQNO on the delta psi, as well as results obtained with a respiration-deficient rho- mutant show that the major component of the diS-C3(3)-reported membrane potential is the delta psi formed on the plasma membrane while mitochondrial potential forms a minor part of the delta psi. Its role may be reflected in the slight depolarisation caused by the F1F0-ATPase inhibitor azide in both rho- mutant and wildtype cells. Blocking the plasma membrane H(+)-ATPase with the DMM-11 inhibitor showed that the enzyme participates in delta psi build-up both in the absence and in the presence of added glucose. Pore-forming agents such as nystatin cause a fast probe entry into the cells signifying membrane damage and extensive binding of the probe to cell constituents reflecting obviously disruption of ionic balance in permeabilised cells. In damaged cells the probe therefore no longer reports on membrane potential but on loss of membrane integrity. The delta psi-independent probe entry signalling membrane damage can be distinguished from the potential-dependent diS-C3(3) uptake into intact cells by being insensitive to the depolarising action of CCCP.
Gásková
D
D
Institute of Physics, Charles University, Prague, Czech Republic. gaskova@karlov.mff.cuni.cz
Brodská
B
B
Holoubek
A
A
Sigler
K
K
D002258:Carbonyl Cyanide m-Chlorophenyl Hydrazone / Q000378:metabolism; D002259:Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone / Q000378:metabolism; D002462:Cell Membrane / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D007476:Ionophores / Q000494:pharmacology; D008564:Membrane Potentials; D008928:Mitochondria / Q000737:chemistry; D009550:Nigericin / Q000494:pharmacology; D009761:Nystatin / Q000494:pharmacology; D012441:Saccharomyces cerevisiae / Q000737:chemistry*; D013050:Spectrometry, Fluorescence; D013997:Time Factors; D014634:Valinomycin / Q000494:pharmacology
D007476:Ionophores; D009761:Nystatin; D014634:Valinomycin; D002259:Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone; D002258:Carbonyl Cyanide m-Chlorophenyl Hydrazone; D009550:Nigericin