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

Publisher Website
Google Scholar
Abstract
Active transport of potassium in K+-starvedNeurospora 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. ForNeurospora, 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 ofNeurospora has been studied with a voltage-clamp technique, using difference-current methods to dissect it from other ion-transport processes in theNeurospora plasma membrane. Addition of 5-200 μM K+ to the bathing medium causes 10–150 mV depolarization of the unclamped membrane, and yields a sigmoidI–V curve with a steep slope (maximal conductance of 10–30 μS/cm2) for voltages of −300 to −100 mV, i.e., in the normal physiologic range. Outside that range the apparentI–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 theI–V difference method under reversing conditions. In the absence of voltage clamping, inhibitors—such as cyanide or vanadate—which block the primary proton pump inNeurospora 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 on the cotransport system itself. Detailed study of the effects of [K+]o and pHo on theI–V curve for K+-H+ symport has revealed that increasing membrane potential systematicallydecreases the apparent affinity of the transporter for K+, butincreases affinity for protons (K m range: for [K+]o, 15–45 μM; 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.