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J. Biochem, 2003, Vol. 134, No. 4 615-623
© 2003 Japanese Biochemical Society

BIOCHEMISTRY

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

Yoichi Nakanishi1, Isamu Yabe2 and Masayoshi Maeshima*,1

1 Laboratory of Cell Dynamics, Graduate School of Bioagricultural Sciences, Nagoya University, Nagoya 464-8601; and 2 the Institute of Molecular and Cellular Biosciences, the University of Tokyo, Tokyo 113-0032

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 x 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.

* To whom correspondence should be addressed. Tel/Fax: +81-52-789-4096, E-mail: maeshima{at}agr.nagoya-u.ac.jp


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