Patch Clamp Analysis of a H+ Pump Heterologously Expressed in Giant Yeast Vacuoles.

Despite the usefulness of the patch-clamp technique, its application to ion pumps and transporters in biomembranes is limited. We developed a novel method for determining the activity of a proton-pumping pyrophosphatase (H+-PPase) made of a single protein. We heterologously highly expressed the enzyme in Saccharomyces cerevisiae, prepared giant vacuoles from the cells, and measured a PPi-dependent electrical current of 18 pA (10.5 fA/µm2) using the patch-clamp technique in the whole-vacuole recording mode. We determined the inhibitor sensitivity and affinity for substrate (Km, 4.6 µM). The enzyme number in a giant vacuole (4.2 × 106) and the molecular activity of the expressed H+-PPase (14 s–1) were determined. An uncoupling-type H+-PPase mutant, of which the 263rd glutamate residue was replaced by aspartate, and of which H+ pump activity was not detected with the fluorescence quenching method, showed a weak current with a high Km. The high accuracy, effectiveness and applicability of the method for exogenously expressed ion transporters were also discussed. [ABSTRACT FROM PUBLISHER]