Potassium-proton symport in Neurospora: kinetic control by pH and membrane potential

Abstract

Active transport of potassium in K+-starved Neurospora was previously shown to resemble closely potassium uptake in yeast, Chlorella, and higher plants, for which K+ pumps or K+/H+-ATPases had been proposed. For Neurospora, however, potassium-proton cotransport was demonstrated to operate, with a coupling ratio of 1 H+ to 1 K+ taken inward so that K+, but not H+, moves against its electrochemical gradient (Rodriguez-Navarro et al., J. Gen. Physiol. 87:649-674). In the present experiments, the current-voltage (I-V) characteristic of K+-H+ cotransport in spherical cells of Neurospora has been studied with a voltage-clamp technique, using difference-current methods to dissect it from other ion-transport processes in the Neurospora plasma membrane. Addition of 5-200 microM K+ to the bathing medium causes 10-150 mV depolarization of the unclamped membrane, and yields a sigmoid I-V curve with a steep slope (maximal conductance of 10-30 microS/cm2) for voltages of -300 to -100 mV, i.e., in the normal physiologic range. Outside that range the apparent I-V curve of the K+-H+ symport saturates for both hyperpolarization and depolarization. It fails to cross the voltage axis at its predicted reversal potential, however, an effect which can be attributed to failure of the I-V difference method under reversing conditions. In the absence of voltage clamping, inhibitors-such as cyanide or vanadate-which block the primary proton pump in Neurospora also promptly inhibit K+ transport and K+-H+ currents. But when voltage clamping is used to offset the depolarizing effects of pump blockade, the inhibitors have no immediate effect on K+-H+ currents. Thus, the inhibition of K+ transport usually observed with these agents reflects the kinetic effect of membrane depolarization rather than any direct chemical action or the cotransport system itself. Detailed study of the effects of [K+]o and pHo on the I-V curve for K+-H+ symport has revealed that increasing membrane potential systematically decreases the apparent affinity of the transporter for K+, but increases affinity for protons (Km range: for [K+]o, 15-45 microM; for [H+]o, 10-35 nM). This behavior is consistent with two distinct reaction-kinetic models, in which (i) a neutral carrier binds K+ first and H+ last in the forward direction of transport, or (ii) a negatively charged carrier (-2) binds H+ first and K+ last.