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Originally published In Press as doi:10.1074/jbc.M001010200 on June 23, 2000

J. Biol. Chem., Vol. 275, Issue 39, 30046-30049, September 29, 2000
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A Novel Method for Measurement of Submembrane ATP Concentration*

Fiona M. GribbleDagger §, Gildas Loussouarn||, Stephen J. TuckerDagger **, Chao ZhaoDagger , Colin G. Nichols, and Frances M. AshcroftDagger Dagger Dagger

From the Dagger  University Laboratory of Physiology, Oxford University, Parks Road, Oxford OX1 3PT, United Kingdom and the  Department of Cell Biology and Physiology, Washington University School of Medicine, St. Louis, Missouri 63110

There has been considerable debate as to whether adenosine triphosphate (ATP) is compartmentalized within cells and, in particular, whether the ATP concentration directly beneath the plasma membrane, experienced by membrane proteins, is the same as that of the bulk cytoplasm. This issue has been difficult to address because there is no indicator of cytosolic ATP, such as those available for Ca2+, capable of resolving the submembrane ATP concentration ([ATP]sm) in real time within a single cell. We show here that mutant ATP-sensitive K+ channels can be used to measure [ATP]sm by comparing the increase in current amplitude on patch excision with the ATP dose-response curve. In Xenopus oocytes, [ATP]sm was 4.6 ± 0.3 mM (n = 29) under resting conditions, slightly higher than that measured for the bulk cytoplasm (2.3 mM). In mammalian (COSm6) cells, [ATP]sm was slightly lower and averaged 1.4 ± 0.1 mM (n = 66). Metabolic poisoning (10 min of 3 mM azide) produced a significant fall in [ATP]sm in both types of cells: to 1.2 ± 0.1 mM (n = 24) in oocytes and 0.8 ± 0.11 mM for COSm6 cells. We conclude that [ATP]sm lies in the low millimolar range and that there is no gradient between bulk cytosolic and submembrane [ATP].

* This work was supported by the Wellcome Trust and by National Institutes of Health Grant HL45742 (to C. G. N.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§ Recipient of a Wellcome Trust Advanced Fellowship for Medical Graduates.

|| Recipient of a fellowship from the American Heart Foundation (Missouri affiliate).

** Recipient of a Wellcome Trust Career Development Award.

Dagger Dagger To whom correspondence should be addressed: University Laboratory of Physiology, Oxford University, Parks Rd., Oxford OX1 3PT, UK. Tel.: 01865-272478; Fax: 01865-272469; E-mail: frances.ashcroft@physiol.ox.ac.uk.

Copyright © 2000 by The American Society for Biochemistry and Molecular Biology, Inc.
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