Effects of inhibitors of ion-motive ATPases on the plasma membrane potential of murine erythroleukemia cells

The membrane electric effects of N,N'-dicyclohexyl-carbodiimide (DCCD) and vanadate were studied in murine erythroleukemia cells (MELC), comparing the patch-clamp technique and the accumulation ratio (ARexp) of 3H]-tetraphenylphosphonium (TPP+). Electrophysiological measurements showed that both these inhibitors produce, at micromolar concentrations, a 20-30 mV hyperpolarization of resting potential (delta psi p) of MELC, which is abolished when the electrochemical equilibrium potential of K+ (EK) is brought close to zero. DCCD and vanadate turned out to have distinct targets on the plasma membrane of MELC (an H+ pump and the Na+,K(+)-ATPase, respectively). Measurements of ARexp showed that: (i) patch-clamp measurements of delta psi p were equivalent to those based on ARexp of antimycin-pretreated cells (ARANT); (ii) DCCD produced a strong increase in ARANT, that was antagonized by carbonyl cyanide p-trifluoromethoxyphenyl-hydrazone (FCCP) and diethylstilbestrol (DES); (iii) vanadate determined a marked increase in ARANT that was insensitive to FCCP, but antagonized by ouabain; (iv) incubation in high K+ medium (HK) brought ARANT to 1.0 in the controls, but did not lower this ratio below 3.0 in the presence of DCCD or vanadate; (v) the total amount of TPP+ taken up by the cells was in any case water extractable by a freezing and thawing procedure. On the whole, our data indicate that DCCD and vanadate hyperpolarize the MELC by increasing the K+ conductance and, at the same time, enhance the TPP+ binding, probably by changing the electrostatic potential profile of the plasma membrane. These effects seem to involve functional modifications of the target pumps, apparently related to the ion-occluding state of these enzymes.