Novel method for measurement of submembrane ATP concentration

There has been considerable debate as to whether adenosine triphosphate (AT P) is compartmentalized within cells and, in particular, whether the ATP co ncentration directly beneath the plasma membrane, experienced by membrane p roteins, is the same as that of the bulk cytoplasm. This issue has been dif ficult to address because there is no indicator of cytosolic ATP, such as t hose available for Ca2+, capable of resolving the submembrane ATP concentra tion ([ATP](sm)) in real time within a single cell. We show here that mutan t ATP-sensitive K+ channels can be used to measure [ATP](sm) by comparing t he increase in current amplitude on patch excision with the ATP dose-respon se curve. In Xenopus oocytes, [ATP](sm) was 4.6 +/- 0.3 mM (n = 29) under r esting conditions, slightly higher than that measured for the bulk cytoplas m (2.3 mM). In mammalian (COSm6) cells, [ATP](sm) was slightly lower and av eraged 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 conclud e that [ATP](sm) lies in the low millimolar range and that there is no grad ient between bulk cytosolic and submembrane [ATP].