Electrogenic active proton pump in Rana esculenta skin and its role in sodium ion transport.

Abstract

Kinetic and electrophysiological studies were carried out in the in vitro Rana esculenta skin, bathed in dilute sodium solution, to characterize the proton pump and coupling between sodium absorption .**GRAPHIC**. and proton excretion .**GRAPHIC**. .**GRAPHIC**. and .**GRAPHIC**. were both dependent on transepithelial potential (.psi.ms); hyperpolarizing the skin decreased .**GRAPHIC**. and increased .**GRAPHIC**. depolarization produced the opposite effects. Amiloride (5 .times. 10-5 M) at a clamped .psi.ms of + 50 mV inhibited .**GRAPHIC**. without affecting .**GRAPHIC**. Variations of .psi.ms or pH had identical effects on .**GRAPHIC**. Ethoxzolamide inhibited .**GRAPHIC**. and simultaneously increased .psi.ms by 15-30 mV. These changes were accompanied by depolarization of the apical membrane potential .psi.mc from -47 to -25 mV and an increase in apical membrane resistance of 30%; no significant effects on basolateral membrane potential (.psi.cs) and resistance (Rb) nor the apical membrane. The proton pump was also inhibited by deoxygenation, oligomycin, dicyclohexylcarbodiimide and vanadate (100, 78, 83 and 100% inhibition respectively). The variations of .**GRAPHIC**. and of the measured electrical currents were significantly correlated. Evidently there is a primary active proton pump, electrogenic and strictly linked to aerobic metabolism. The current-voltage (I-V) relation of the proton pump was obtained as the difference in the I-V curves of the apical membrane extracted before and after proton-pump inhibition by ethoxzolamide during amiloride block of sodium transport. The proton-pump current (IP) was best-described by a saturable exponential function of .psi.mc. Maximal pump current .**GRAPHIC**. was calculated to be 200 nequiv h-1 cm-2 at a .psi.mc of +50 mV and the pump reversal potential .**GRAPHIC**. was -130 mV. The effect of ethoxzolamide to depolarize .psi.mc was dependent on the relation between .psi.mc and .**GRAPHIC**. Maximal induced depolarization occurred at a .psi.mc of +50 mV whereas ethoxzolamide exerted minimal effect on .psi.mc when the .**GRAPHIC**. was approached either by voltage clamping the apical membrane or by the addition of amiloride. Electroneutral sodium-proton countertransport is not the mechanism of active proton excretion in frog skin but that it is the proton excretion which provides a favorable electrical driving force for passive apical sodium entry. the apparent coupling between net proton and sodium fluxes depends on the relation between .psi.mc and .**GRAPHIC**. and the effect of the sodium-electrode-like behavior of the apical membrane on this relation.