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Biophysical Journal 85:3730-3738 (2003)
© 2003 The Biophysical Society

Electrophysiological Analysis of the Yeast V-Type Proton Pump: Variable Coupling Ratio and Proton Shunt

Carsten Kettner *, Adam Bertl *, Gerhard Obermeyer {dagger}, Clifford Slayman {ddagger} and Hermann Bihler *

* Botanisches Institut I, Universität Karlsruhe, Karlsruhe, Germany; {dagger} Institut für Pflanzenphysiologie, Universität Salzburg, Salzburg, Austria; and {ddagger} Department of Cellular and Molecular Physiology, Yale School of Medicine, New Haven, Connecticut

Correspondence: Address reprint requests to Hermann Bihler, Botanisches Institut I, Universität Karlsruhe, Kaiserstrasse 12, 76128 Karlsruhe, Germany. Tel.: 49-721-608-6887; Fax: 49-721-608-4193; E-mail: db49{at}rz.uni-karlsruhe.de.

Isolated vacuoles from the yeast Saccharomyces cerevisiae were examined in the whole-vacuole mode of patch recording, to get a detailed functional description of the vacuolar proton pump, the V-ATPase. Functioning of the V-ATPase was characterized by its current-voltage (I-V) relationship, obtained for various levels of vacuolar and cytosolic pH. I-V curves for the V-ATPase were computed as the difference between I-V curves obtained with the pump switched on (ATP, ADP, and Pi present) or off (no ATP). These difference current-voltage relationships usually crossed the voltage axis within the experimental range (from -80 to +80 mV), thus measuring the reversal voltage (ER) for the V-ATPase, which could be compared with the standing ion gradients and free energy of ATP hydrolysis, to calculate the apparent pump stoichiometry or coupling ratio: the number of protons transported for each ATP molecule hydrolyzed. This ratio was found to depend strongly upon the pH difference ({Delta}pH) across the vacuolar membrane, being ~2H+/ATP at high {Delta}pH (4 pH units) and increasing to >4H+/ATP for small or zero {Delta}pH. That result is in quantitative agreement with previous determinations on plant vacuoles. Considerations of purely electrical behavior, together with the physical properties of a recent detailed structural model for V-ATPases, led to a linear equivalent circuit—which quantitatively accounts for all observations of variable coupling ratios in fungal and plant V-ATPases by variations of the conductance for bona fide proton pumping (GP) through the ATPase relative to independent proton shunting (GS) through the same protein.




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