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A Novel Method for Measurement of Submembrane ATP Concentration

A Novel Method for Measurement of Submembrane ATP Concentration THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 275, No. 39, Issue of September 29, pp. 30046 –30049, 2000 © 2000 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A. A Novel Method for Measurement of Submembrane ATP Concentration* Received for publication, February 8, 2000, and in revised form, May 8, 2000 Published, JBC Papers in Press, June 23, 2000, DOI 10.1074/jbc.M001010200 Fiona M. Gribble‡§, Gildas Loussouarn¶i, Stephen J. Tucker‡**, Chao Zhao‡, Colin G. Nichols¶, and Frances M. Ashcroft‡ ‡‡ From the ‡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 been made for liver cells under conditions of metabolic inhibi- adenosine triphosphate (ATP) is compartmentalized tion (3, 4). By contrast, studies of the Na/K-ATPase in eryth- within cells and, in particular, whether the ATP concen- rocytes have suggested that [ATP] may actually be higher sm tration directly beneath the plasma membrane, experi- than that of the bulk cytosol (4, 5). Resolution of this issue enced by membrane proteins, is the same as that of the requires a method for measuring [ATP] . sm bulk cytoplasm. This issue has been difficult to address One way to measure [ATP] would be to use an ATP-sensitive sm 21 1 because there is no indicator of cytosolic ATP, such as channel as a biosensor, in the same way that Ca -activated K those available for Ca , capable of resolving the sub- channels have been used to monitor submembrane [Ca ] (6). membrane ATP concentration ([ATP] ) in real time sm The wild-type K channel is not suitable for this purpose ATP within a single cell. We show here that mutant ATP- because in intact cells the inhibitory effect of ATP is partially sensitive K channels can be used to measure [ATP] sm masked by an additional stimulatory action of MgADP (7, 8). by comparing the increase in current amplitude on However, the K channel is an octamer of two subunits: Kir6.2 ATP patch excision with the ATP dose-response curve. In and SUR (9, 10). Kir6.2 is an inwardly rectifying potassium Xenopus oocytes, [ATP] was 4.6 6 0.3 mM (n 5 29) sm channel pore that is intrinsically sensitive to ATP, whereas SUR under resting conditions, slightly higher than that endows Kir6.2 with sensitivity to the stimulatory effects of M). In mamma- measured for the bulk cytoplasm (2.3 m MgADP (11–14). Although wild-type K channels require both ATP lian (COSm6) cells, [ATP] was slightly lower and av- sm subunits for functional activity, a mutant form of Kir6.2 with a M (n 5 66). Metabolic poisoning (10 min eraged 1.4 6 0.1 m C-terminal truncation of 26 or 36 amino acids (Kir6.2DC) is M azide) produced a significant fall in [ATP] of3m in sm capable of independent functional expression (14). This channel both types of cells: to 1.2 6 0.1 mM (n 5 24) in oocytes and M for COSm6 cells. We conclude that [ATP] is inhibited by ATP but is not activated by MgADP, making it a 0.8 6 0.11 m sm lies in the low millimolar range and that there is no gra- potential tool for monitoring [ATP] . sm dient between bulk cytosolic and submembrane [ATP]. In this paper, we show that Kir6.2DC can be used as a biosensor for measurement of [ATP] . We found that in Xeno- sm pus oocytes [ATP] was ;5mM under resting conditions, sm ATP-sensitive potassium channels (K channels) couple whereas in mammalian cells, [ATP] averaged around 1 mM. ATP sm cell metabolism to electrical activity and play important roles Metabolic poisoning produced a significant fall in [ATP] in both sm in the physiology and pathophysiology of many tissues (1). In types of cells. Thus, [ATP] is similar to or greater than the sm pancreatic b-cells these channels couple changes in blood glu- average [ATP] , and submembrane ATP gradients are unlikely cyt cose concentration to insulin secretion. In cardiac tissue they to contribute to the metabolic regulation of K channel activity. ATP are involved in action potential shortening during ischemia, EXPERIMENTAL PROCEDURES and in vascular smooth muscle they regulate vessel tone. Al- Oocyte Experiments—A C-terminal truncation of 26 or 36 amino though K channels are inhibited by intracellular ATP with ATP TM acids of mouse Kir6.2 (GenBank accession number D50581) was a K of ;10 mM in excised patches, substantial channel activity made by the introduction of a stop codon at the appropriate residue by is observed in intact b-cells under conditions where measured site-directed mutagenesis. There was no difference in the ATP sensi- cytosolic [ATP] is 3–5 mM. It has therefore been proposed that tivity of mutants carrying a 26- or 36-amino acid truncation (14), so they are simply referred to here as Kir6.2DC, for simplicity. Site- the submembrane ATP concentration ([ATP] ) may be lower sm directed mutations in Kir6.2DC were made using the pALTER vector. than that of the bulk cytoplasm (1, 2). A similar suggestion has Synthesis of mRNA encoding wild-type and mutant mouse Kir6.2 was carried out as described previously (15). * This work was supported by the Wellcome Trust and by National Xenopus oocytes were defolliculated and injected with ;2ngof Institutes of Health Grant HL45742 (to C. G. N.). The costs of publica- mRNA encoding either wild-type or mutated Kir6.2DC, or Kir1.1a. The tion of this article were defrayed in part by the payment of page final injection volume was ;50 nl/oocyte. Isolated oocytes were main- charges. This article must therefore be hereby marked “advertisement” tained in tissue culture and studied 1– 4 days after injection (15). in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Whole-cell currents were measured at 18 –24 °C using a standard 2- § Recipient of a Wellcome Trust Advanced Fellowship for Medical electrode voltage clamp (14) in (mM): 90 KCl, 1 MgCl , 1.8 CaCl ,5 2 2 Graduates. HEPES (pH 7.4 with KOH). The holding potential was 210 mV. Cur- i Recipient of a fellowship from the American Heart Foundation (Mis- rents were filtered at 1 kHz, digitized at 4 kHz, and measured 280 –295 souri affiliate). ms after the start of the voltage pulse. ** Recipient of a Wellcome Trust Career Development Award. Macroscopic currents were recorded from giant inside-out patches at ‡‡ To whom correspondence should be addressed: University Labora- a holding potential of 0 mV and at 18 –24 °C (15). The pipette solution tory of Physiology, Oxford University, Parks Rd., Oxford OX1 3PT, UK. contained (mM): 140 KCl, 1.2 MgCl , 2.6 CaCl , 10 HEPES (pH 7.4 with Tel.: 01865-272478; Fax: 01865-272469; E-mail: frances.ashcroft@ 2 2 physiol.ox.ac.uk. KOH). The internal (bath) solution contained (mM): 110 KCl, 1.4 The abbreviations used are: [ATP] , submembrane ATP concentra- MgCl , 30 KOH, 10 EGTA, 10 HEPES (pH 7.2 with KOH), and nucle- sm tion; Ap A, diadenosine polyphosphate. otides as indicated. Macroscopic currents were recorded in response to 30046 This paper is available on line at http://www.jbc.org This is an Open Access article under the CC BY license. A Submembrane ATP Sensor 30047 FIG.1. A, macroscopic currents recorded from a giant patch on an oocyte injected with Kir6.2DC. The holding potential was 0 mV, and the voltage was successively ramped from 2110 to 1100 mV over a 3-s period. The patch was excised into ATP-free solution at the arrow.1mM ATP was subsequently added to the intracellular solution as indicated. FIG.2. A, macroscopic currents recorded from four different patches B, mean dose-response relationships for wild-type Kir6.2DC(n 5 11) in response to a series of voltage ramps from 2110 to 1100 mV. Oocytes and for Kir6.2DC-I167M (n 5 5), Kir6.2DC-R50G (n 5 6), and Kir6.2DC- were injected with mRNAs encoding Kir6.2DC, Kir6.2DC-E179Q, E179Q (n 5 7). The slope conductance (G) is given as a fraction of the Kir6.2DC-E167M, or Kir6.2DC-R50G. The patch was excised into ATP- mean conductance (G ) obtained in control solution before and after free solution at the arrows. The dashed line indicates the zero current exposure to ATP. The lines are the best fit of the Hill equation to the level. B, relationship between the increase in conductance on patch exci- data using the mean values for K given in the text. sion and 1/K for channel inhibition by ATP for the channels indicated. The conductance following excision is expressed as a fraction of that in the 3-s voltage ramps from 2110 to 1100 mV, filtered at 0.2 kHz, and cell-attached configuration. The line is drawn to the equation G/G 5 1/(1 sampled at 0.5 kHz. The slope conductance was measured by fitting a 1 ([ATP]/K ) ) with h 5 1.0 and the concentration of ATP 5 5.7 mM. straight line to the data between 220 and 2100 mV; the average of five consecutive ramps was calculated in each solution, except when the tions contained (mM): 140 KCl, 1 K-EGTA, 10 K-HEPES (pH 7.3), with currents were measured immediately after patch excision, when only 2 additions as described. ramps were averaged to avoid errors due to channel rundown. Currents Data Analysis—All data are given as mean 6 S.E. The symbols in the were corrected for leak (,1% of the total current) by subtraction of the figures indicate the mean, and the vertical bars indicate one S.E. (where mean current recorded in Kir6.2DC-injected oocytes at a maximally this is larger than the symbol). ATP dose-response relationships were effective concentration of ATP. fit to the Hill equation: G/G 5 1/(1 1 ([ATP]/K ) )), where G is the slope COSm6 Cell Experiments—Mouse Kir6.2 and hamster SUR1 (Gen- c i TM conductance in the presence of ATP, G is the mean conductance ob- Bank accession number L40623) were used for these experiments. c tained in control solution (before and after ATP application), [ATP] is Point mutations were made in a Kir6.2DC36-C166S construct the ATP concentration, K is the ATP concentration at which inhibition (Kir6.2DCS), in which the C terminus was truncated by 36 amino acids i is half-maximal, and h is the slope factor (Hill coefficient). Statistical and cysteine 166 was replaced by serine. Mutations were prepared by significance was tested using Student’s t test. overlap extension at the junctions of relevant residues using the sequen- tial polymerase chain reaction (16), and the resulting polymerase chain RESULTS reaction products were subcloned into the pCMV6b vector for transfec- Oocyte Experiments—Currents recorded from cell-attached tion. In some experiments, we used a mutant form of SUR1 in which glycine 1485 was replaced by aspartic acid (abbreviated here as patches on oocytes injected with Kir6.2DC were very small but SUR1-GD). increased ;50-fold following patch excision (Fig. 1A). This in- COSm6 cells (a variety of COS cells) were plated at a density of crease in conductance was not observed in water-injected oo- ;2.5 3 10 cells per well in 30-mm 6-well dishes and cultured in cytes, indicating that it results from activation of Kir6.2DC Dulbecco’s modified Eagle’s medium plus 10 mM glucose supplemented currents that are inhibited in the cell-attached configuration. with fetal calf serum (10%), penicillin (100 units/ml), and streptomycin Subsequent application of 1 mM ATP to the intracellular mem- (100 mg/ml). The next day, cells were transfected with pCMV6b- Kir6.2DCS (with mutations as described), pECE-SUR1-GD, and brane surface largely blocked Kir6.2DC currents, suggesting pGreenLantern (Life Technologies, Inc.) using LipofectAMINE reagent that the submembrane ATP concentration may be responsible (Life Technologies, Inc.) according to the manufacturer’s instructions. for channel inhibition in the intact oocyte. Consistent with this Currents were recorded at 250 mV from cell-attached and inside-out idea, large currents were recorded from cell-attached patches patches from green fluorescent cells using a chamber that allowed rapid on oocytes expressing the ATP-insensitive Kir channel Kir1.1a, solution changes (16). Current amplitudes were measured by fitting a and no change in conductance was observed on patch excision. straight line through the data points once the current had reached a steady state. Both bath (intracellular) and pipette (extracellular) solu- The mean Kir1.1a conductance was 15.0 6 3.6 nanosiemens in 30048 A Submembrane ATP Sensor TABLE I Estimation of [ATP] from the increase in conductance following sm patch excision 1/h [ATP] was calculated from [ATP] 5 (G /G 2 1) 3 K , where G is 2 1 i 1 the slope conductance in the cell-attached condition, G is the slope conductance immediately after excision of the same patch, K is the concentration of ATP required to block the channel half-maximally, and h is the Hill coefficient for the dose-response curve for ATP inhibition. Oocytes Clone G G G /G [ATP] n 1 2 2 1 sm nanosiemens mM Kir6.2DC 0.56 6 0.05 28 6251 6 2 5.8 6 0.3 9 Kir6.2DC-E179Q 2.2 6 0.7 41 6922 6 4 5.0 6 0.8 8 Kir6.2DC-I167M 2.6 6 0.8 16.4 6 4.4 7.2 6 0.9 3.2 6 0.5 8 Kir6.2DC-R50G 2.3 6 0.7 5.8 6 1.9 2.4 6 0.2 4.2 6 0.5 4 COSm6 cells Clone G /G [ATP] n 2 1 sm mM Kir6.2DCS-I154C 0.15 6 0.06 1.8 6 0.6 4 Kir6.2DCS 1 SUR1-GD 0.47 6 0.06 0.96 6 0.14 29 Kir6.2DCS-I154C 1 SUR1-GD 0.12 6 0.04 1.4 6 0.3 14 Kir6.2DCS-T171C 1 SUR1-GD 0.65 6 0.10 1.83 6 0.28 19 the cell-attached patch and 15.5 6 3.9 nanosiemens after patch excision (n 5 5, data not shown). The mean conductance in control (water-injected) oocytes was ,0.2 nanosiemens. A critical test of whether the amplitude of Kir6.2DC currents in the intact cell reflects [ATP] is to examine mutant chan- sm nels with altered ATP sensitivity, because these should pro- duce markedly different current amplitudes in cell-attached patches yet yield similar values of [ATP] . We studied three sm different Kir6.2DC mutations that reduce the channel ATP sensitivity to differing extents: R50G, I167M, and E179Q (Fig. 1B). Half-maximal inhibition (K ) of wild-type Kir6.2DC cur- rents was produced by 115 6 6 mM ATP (n 5 11). Mutation of the arginine at position 50 to glycine (R50G) reduced the K for ATP inhibition to 3.4 mM, mutation of isoleucine 167 to methi- onine (I167M) decreased the K to 640 mM, and mutation of FIG.3. Macroscopic currents recorded from four different glutamate 179 to glutamine (E179Q) reduced the K to 300 mM i membrane patches on COSm6 cells transfected with Kir6.2DCS- (17). As expected, channels that showed lower ATP sensitivity I154C, Kir6.2DCS-I154C 1 SUR1-GD, Kir6.2DCS 1 SUR1-GD, or Kir6.2DCS-T171C 1 SUR1-GD. The holding potential was 250 mV. exhibited larger currents in the cell-attached configuration and The patch was excised into ATP-free solution at the arrows. ATP (10 a smaller increase in conductance on patch excision (Fig. 2A mM,1mM, or 0.1 mM) was subsequently added to the intracellular and Table I). There was a reciprocal relationship between the solution as indicated. The dashed line indicates the zero current level. K for current inhibition by ATP and the increase in conduct- ance on patch excision (Fig. 2B), and the [ATP] calculated rents (8, 18) such that coexpressed channels should only be sm from each set of data was similar (see Fig. 4 and Table I). This sensitive to [ATP]. As shown in Fig. 4 and Table I, coexpression constitutes good evidence that the principal cytosolic regulator of SUR1-GD with Kir6.2DCS-I154C does not significantly alter of Kir6.2DC activity is [ATP] . The mean value of the sub- the estimated [ATP] (1.4 6 0.3 mM versus 1.8 6 0.6 mM). sm sm membrane ATP concentration in oocytes calculated from all the We also examined additional Kir6.2DCS mutants that dis- data given in Table I (and Fig. 2) is 4.6 6 0.3 mM (n 5 29) and played a range of ATP sensitivities. For each patch the mem- that from the slope of the graph in Fig. 2B is 5.7 mM. brane current before and immediately after excision was meas- Mammalian Cell Experiments—We also measured the sub- ured, and the ATP dose-response curve was then constructed. membrane ATP concentration in mammalian cells using a sim- The K for ATP inhibition of Kir6.2DCS-I154C 1 SUR1-GD, ilar approach with another C-terminally deleted construct of Kir6.2DCS 1 SUR1-GD, and Kir6.2DCS-T171C 1 SUR1-GD Kir6.2 (Kir6.2DCS-I154C). This contains the double mutation currents were 0.49 6 0.1 (n 5 14), 0.78 6 0.09 (n 5 29), and C166S and I154C, which shifts the ATP sensitivity of the 3.09 6 0.49 mM (n 5 19), respectively. As observed in the oocyte channel into the appropriate physiological range: K 5 0.54 6 experiments, those channels that were least sensitive to ATP 0.12 mM (n 5 4). Table I shows that the estimated [ATP] in exhibited the largest currents in the cell-attached configura- sm COSm6 cells using Kir6.2DCS-I154C was 1.8 1 0.6 mM. The tion and the smallest increment in current on patch excision patch current level in such experiments was generally quite (Fig. 3). The value of [ATP] estimated for the different K sm ATP low. As reported previously (14), the level of current is en- channel mutants varied between 1 and 1.8 mM (Fig. 4 and hanced by coexpression of SUR1. To obtain larger patch cur- Table I), with an overall mean value of 1.36 6 0.13 mM (n 5 66). rents, and hence increase the accuracy of measured channel These values are somewhat lower than those found in oocytes, activities, we performed further experiments on cells coex- suggesting that [ATP] may be lower in COSm6 cells. sm pressing Kir6.2DCS constructs with a mutant SUR1 containing Effects of Metabolic Inhibition—In intact Xenopus oocytes, the point mutation G1485D (SUR1-GD). This mutation abol- inhibition of cell metabolism by 3 mM azide leads to an increase ishes nucleotide diphosphate stimulation of K channel cur- in the whole-cell K current (15). Using the value for resting ATP ATP A Submembrane ATP Sensor 30049 ATP from certain compartments (such as the yolk platelets), or it may indicate an inverse ATP gradient between the mem- brane and bulk cytosol. We favor the former explanation be- cause a similar difference was found in oocytes of the frog Rana pipiens in biochemical measurements of cytosolic (6 mM) and total (2.5 mM) [ATP] (24). Whatever the reason, our results do not support the widely held view that the submembrane ATP concentration is lower than that of the bulk cytosol (1– 4). Recently, the submembrane ATP concentration has been esti- mated in pancreatic b-cells using a membrane-tagged luciferin and found to be ;1mM under resting conditions (3 mM glucose) (25). Our results on mammalian cells are in good agreement with these studies. Furthermore, the fact that two different methods provide a similar value for [ATP] helps validate the different sm assumptions intrinsic to each method. The bulk cytosolic [ATP] FIG.4. Mean calculated ATP concentrations for oocytes (left) in various mammalian cells is also ;1mM: 1.0 mM in b-cells (25), or COSm6 cells (right) expressing the indicated K channel ATP 0.9 mM in HeLa cells (26), and 0.8 –1.2 mM in COS-1 cells (27). constructs in the presence and absence of metabolic inhibitor. The advantage of using Kir6.2DC as a biosensor is that it is able Black bars indicate the ATP concentration in control solution. Gray bars indicate the ATP concentration in the presence of the metabolic to respond very rapidly to changes in [ATP] . sm inhibitor azide (3 mM). In conclusion, our results suggest that Kir6.2DC alone, or Kir6.2 coexpressed with a mutant SUR1, may be used as tools to monitor the submembrane ATP concentration in real time in [ATP] obtained above, we can estimate the fall in submem- sm a single cell. Inside-out patches excised from cells expressing brane [ATP] following metabolic inhibition. Fig. 4 shows the mutant K channels might also be used to detect the local estimated [ATP] in the presence of 3 mM azide. The mean ATP sm release of ATP from purinergic neurons, as described for ace- [ATP] , calculated from all data, fell to 1.2 6 0.1 mM (n 5 24) sm tylcholine release using acetylcholine receptor channels (28). In when metabolism was inhibited. This value is similar to that a similar fashion, inside-out macropatches containing mutant measured biochemically for the bulk cytoplasm in the presence K channels might serve to monitor exocytosis from single of3mM azide (1.7 mM, Ref. 15). ATP secretory cells, as many types of secretory granules contain ATP. Submembrane [ATP] also fell in mammalian cells incubated Importantly, our data also demonstrate that the submembrane in3mM azide (Fig. 4) to a mean value of 0.76 6 0.11 mM (n 5 ATP concentration lies in the millimolar, rather than the micro- 12) after 10 min (averaged for all Kir6.2DCS mutants coex- molar, range in both Xenopus oocytes and mammalian cells. pressed with SUR1-GD). Even greater falls in [ATP] were sm observed when cells were preincubated in the presence of 1 mM REFERENCES 2-deoxyglucose and 2.5 mg/ml oligomycin (Fig. 4); in this case, the 1. Ashcroft, F. M., and Ashcroft, S. J. H. (1990) Cell. Signalling 2, 197–214 2. Niki, I., Ashcroft, F. M., and Ashcroft, S. J. H. (1989) FEBS Lett. 257, 361–364 mean value of [ATP] was 0.1 6 0.06 mM (n 5 5) after a 30-min sm 3. Aw, T. Y., and Jones, D. P. (1985) Am. J. Physiol. 249, C385–C392 preincubation (measured for Kir6.2DCS mutants coexpressed 4. Jones, D. P. (1985) Am. J. Physiol. 250, C663–C675 with SUR1-GD). This is consistent with earlier studies that have 5. Proverbio, F., and Hoffman, J. F. (1977) J. Gen. Physiol. 69, 605– 632 6. Prakriya, M., Solaro, C. R., and Lingle, C. J. (1996) J. Neurosci. 16, 4344 – 4359 shown that this protocol induces a marked activation of wild-type 7. Kakei, M., Kelly, R. P., Ashcroft, S. J. H., and Ashcroft, F. M. (1986) FEBS Lett. K channel activity, as measured by rubidium efflux (18). ATP 208, 63– 66 8. Nichols, C. G., Shyng, S. L., Nestorowicz, A., Glaser, B., Clement, J. P., IV, DISCUSSION Gonzalez, G., Aguilar-Bryan, L., Permutt, M. A., and Bryan, J. (1996) Science 272, 1785–1787 It is implicit in the use of Kir6.2DC to measure [ATP] that sm 9. Clement, J. P., IV, Kunjilwar, K., Gonzalez, G., Schwanstecher, M., Panten, other cytosolic factors have little or no effect on channel activity U., Aguilar-Bryan, L., and Bryan, J. (1997) Neuron 18, 827– 838 or ATP sensitivity. Our results support this idea, because a 10. Shyng, S. L., and Nichols, C. G. (1997) J. Gen. Physiol. 110, 655– 664 11. Inagaki, N., Gonoi, T., Clement, J. P., IV, Namba, N., Inazawa, J., Gonzalez, similar estimate of [ATP] is obtained for channels with differ- sm G., Aguilar-Bryan, L., Seino, S., and Bryan, J. (1995) Science 270, ent K for ATP inhibition. We have shown elsewhere that 1166 –1169 12. Sakura, H., Amma ¨la ¨ , C., Smith, P. A., Gribble, F. M., and Ashcroft, F. M. Kir6.2DC is selectively inhibited by ATP and is not substantially (1995) FEBS Lett. 377, 338 –344 blocked by other nucleoside triphosphates (K . 5mM, Ref. 17). 13. Inagaki, N., Gonoi, T., Clement, J. P., IV, Wang, C. Z., Aguilar-Bryan, L., Only ATP, Ap A(K 5;100 mM), and ADP (K 5;250 mM) were Bryan, J., and Seino, S. (1996) Neuron 16, 1011–1017 4 i i 14. Tucker, S. J., Gribble, F. M., Zhao, C., Trapp, S., and Ashcroft, F. M. (1997) potent blockers of Kir6.2DC. The concentrations of Ap A and Nature 387, 179 –183 ADP in the bulk cytoplasm are believed to lie within the low and 15. Gribble, F. M., Ashfield, R., Amma ¨la ¨ , C., and Ashcroft, F. M. (1997) J. Physiol. intermediate micromolar range, respectively (19, 20), and are 498, 87–98 16. Koster, J. C., Sha, Q., and Nichols, C. G. (1999) J. Gen. Physiol. 4, 203–213 much lower than that of ATP. Thus we assume that the activity 17. Tucker, S. J., Gribble, F. M., Proks, P., Trapp, S., Ryder, T. J., Haug, T., of Kir6.2DC in the intact cell is largely determined by [ATP] . Reimann, F., and Ashcroft, F. M. (1998) EMBO J. 17, 3290 –3296 sm 18. Shyng, S. L., Ferrigni, T., and Nichols, C. G. (1997) J. Gen. Physiol. 110, Recent studies have shown that the membrane phospholipid 643– 654 phosphatidylinositol bisphosphate decreases the sensitivity of 19. Ripoll, C., Martin, F., Manuel-Rovira, J., Pintor, J., Miras-Portugal, M. T., and the K channel to ATP and that the gradual loss of phos- Soria, B. (1996) Diabetes 45, 1431–1434 ATP 20. Ghosh, A., Ronner, P., Cheong, E., Khalid, P., and Matschinsky, F. M. (1991) phatidylinositol bisphosphate from the membrane that occurs J. Biol. Chem. 266, 22887–22892 following patch excision is accompanied by an enhanced ATP 21. Fan, Z., and Makielski, J. C. (1997) J. Biol. Chem. 272, 5388 –5395 22. Shyng, S.-L., and Nichols, C. G. (1998) Science 282, 1138 –1141 sensitivity (21–23). Such a lowering of the K for ATP inhibition 23. Baukrowitz, T., Schulte, U., Oliver, D., Herlitze, S., Krauter, T., Tucker, S. J., would lead to an underestimate of [ATP] . Thus to avoid sm Ruppersberg, J. P., and Fakler, B. (1998) Science 282, 1141–1144 artifactual changes in ATP sensitivity, ATP dose-response 24. Miller, D. S., and Horowitz, S. B. (1986) J. Biol. Chem. 261, 13911–13915 25. Kennedy, H. J., Pouli, A. E., Ainscow, E. K., Jouaville, L. S., Rizzuto, R., and curves were measured immediately after patch excision. Rutter, G. A. (1999) J. Biol. Chem. 274, 13281–13291 The mean value of the submembrane ATP concentration in 26. Wang, R. H, Tao, L., Trumbore, M. W., Berger, S. L. (1997) J. Biol. Chem. 272, 26405–26412 oocytes (;5mM) is approximately double that measured from 27. Sippel, C. J., Dawson, P. A., Shen, T., and Perlmutter, D. H. (1997) J. Biol. the whole oocyte using a biochemical method (2.3 mM, Ref. 15). Chem. 272, 18290 –18297 The difference may be only apparent and reflect exclusion of 28. Hume, R. I., Role, L. W., and Fischbach, G. D. (1983) Nature 305, 632– 634 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Journal of Biological Chemistry Unpaywall

A Novel Method for Measurement of Submembrane ATP Concentration

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THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 275, No. 39, Issue of September 29, pp. 30046 –30049, 2000 © 2000 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A. A Novel Method for Measurement of Submembrane ATP Concentration* Received for publication, February 8, 2000, and in revised form, May 8, 2000 Published, JBC Papers in Press, June 23, 2000, DOI 10.1074/jbc.M001010200 Fiona M. Gribble‡§, Gildas Loussouarn¶i, Stephen J. Tucker‡**, Chao Zhao‡, Colin G. Nichols¶, and Frances M. Ashcroft‡ ‡‡ From the ‡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 been made for liver cells under conditions of metabolic inhibi- adenosine triphosphate (ATP) is compartmentalized tion (3, 4). By contrast, studies of the Na/K-ATPase in eryth- within cells and, in particular, whether the ATP concen- rocytes have suggested that [ATP] may actually be higher sm tration directly beneath the plasma membrane, experi- than that of the bulk cytosol (4, 5). Resolution of this issue enced by membrane proteins, is the same as that of the requires a method for measuring [ATP] . sm bulk cytoplasm. This issue has been difficult to address One way to measure [ATP] would be to use an ATP-sensitive sm 21 1 because there is no indicator of cytosolic ATP, such as channel as a biosensor, in the same way that Ca -activated K those available for Ca , capable of resolving the sub- channels have been used to monitor submembrane [Ca ] (6). membrane ATP concentration ([ATP] ) in real time sm The wild-type K channel is not suitable for this purpose ATP within a single cell. We show here that mutant ATP- because in intact cells the inhibitory effect of ATP is partially sensitive K channels can be used to measure [ATP] sm masked by an additional stimulatory action of MgADP (7, 8). by comparing the increase in current amplitude on However, the K channel is an octamer of two subunits: Kir6.2 ATP patch excision with the ATP dose-response curve. In and SUR (9, 10). Kir6.2 is an inwardly rectifying potassium Xenopus oocytes, [ATP] was 4.6 6 0.3 mM (n 5 29) sm channel pore that is intrinsically sensitive to ATP, whereas SUR under resting conditions, slightly higher than that endows Kir6.2 with sensitivity to the stimulatory effects of M). In mamma- measured for the bulk cytoplasm (2.3 m MgADP (11–14). Although wild-type K channels require both ATP lian (COSm6) cells, [ATP] was slightly lower and av- sm subunits for functional activity, a mutant form of Kir6.2 with a M (n 5 66). Metabolic poisoning (10 min eraged 1.4 6 0.1 m C-terminal truncation of 26 or 36 amino acids (Kir6.2DC) is M azide) produced a significant fall in [ATP] of3m in sm capable of independent functional expression (14). This channel both types of cells: to 1.2 6 0.1 mM (n 5 24) in oocytes and M for COSm6 cells. We conclude that [ATP] is inhibited by ATP but is not activated by MgADP, making it a 0.8 6 0.11 m sm lies in the low millimolar range and that there is no gra- potential tool for monitoring [ATP] . sm dient between bulk cytosolic and submembrane [ATP]. In this paper, we show that Kir6.2DC can be used as a biosensor for measurement of [ATP] . We found that in Xeno- sm pus oocytes [ATP] was ;5mM under resting conditions, sm ATP-sensitive potassium channels (K channels) couple whereas in mammalian cells, [ATP] averaged around 1 mM. ATP sm cell metabolism to electrical activity and play important roles Metabolic poisoning produced a significant fall in [ATP] in both sm in the physiology and pathophysiology of many tissues (1). In types of cells. Thus, [ATP] is similar to or greater than the sm pancreatic b-cells these channels couple changes in blood glu- average [ATP] , and submembrane ATP gradients are unlikely cyt cose concentration to insulin secretion. In cardiac tissue they to contribute to the metabolic regulation of K channel activity. ATP are involved in action potential shortening during ischemia, EXPERIMENTAL PROCEDURES and in vascular smooth muscle they regulate vessel tone. Al- Oocyte Experiments—A C-terminal truncation of 26 or 36 amino though K channels are inhibited by intracellular ATP with ATP TM acids of mouse Kir6.2 (GenBank accession number D50581) was a K of ;10 mM in excised patches, substantial channel activity made by the introduction of a stop codon at the appropriate residue by is observed in intact b-cells under conditions where measured site-directed mutagenesis. There was no difference in the ATP sensi- cytosolic [ATP] is 3–5 mM. It has therefore been proposed that tivity of mutants carrying a 26- or 36-amino acid truncation (14), so they are simply referred to here as Kir6.2DC, for simplicity. Site- the submembrane ATP concentration ([ATP] ) may be lower sm directed mutations in Kir6.2DC were made using the pALTER vector. than that of the bulk cytoplasm (1, 2). A similar suggestion has Synthesis of mRNA encoding wild-type and mutant mouse Kir6.2 was carried out as described previously (15). * This work was supported by the Wellcome Trust and by National Xenopus oocytes were defolliculated and injected with ;2ngof Institutes of Health Grant HL45742 (to C. G. N.). The costs of publica- mRNA encoding either wild-type or mutated Kir6.2DC, or Kir1.1a. The tion of this article were defrayed in part by the payment of page final injection volume was ;50 nl/oocyte. Isolated oocytes were main- charges. This article must therefore be hereby marked “advertisement” tained in tissue culture and studied 1– 4 days after injection (15). in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Whole-cell currents were measured at 18 –24 °C using a standard 2- § Recipient of a Wellcome Trust Advanced Fellowship for Medical electrode voltage clamp (14) in (mM): 90 KCl, 1 MgCl , 1.8 CaCl ,5 2 2 Graduates. HEPES (pH 7.4 with KOH). The holding potential was 210 mV. Cur- i Recipient of a fellowship from the American Heart Foundation (Mis- rents were filtered at 1 kHz, digitized at 4 kHz, and measured 280 –295 souri affiliate). ms after the start of the voltage pulse. ** Recipient of a Wellcome Trust Career Development Award. Macroscopic currents were recorded from giant inside-out patches at ‡‡ To whom correspondence should be addressed: University Labora- a holding potential of 0 mV and at 18 –24 °C (15). The pipette solution tory of Physiology, Oxford University, Parks Rd., Oxford OX1 3PT, UK. contained (mM): 140 KCl, 1.2 MgCl , 2.6 CaCl , 10 HEPES (pH 7.4 with Tel.: 01865-272478; Fax: 01865-272469; E-mail: frances.ashcroft@ 2 2 physiol.ox.ac.uk. KOH). The internal (bath) solution contained (mM): 110 KCl, 1.4 The abbreviations used are: [ATP] , submembrane ATP concentra- MgCl , 30 KOH, 10 EGTA, 10 HEPES (pH 7.2 with KOH), and nucle- sm tion; Ap A, diadenosine polyphosphate. otides as indicated. Macroscopic currents were recorded in response to 30046 This paper is available on line at http://www.jbc.org This is an Open Access article under the CC BY license. A Submembrane ATP Sensor 30047 FIG.1. A, macroscopic currents recorded from a giant patch on an oocyte injected with Kir6.2DC. The holding potential was 0 mV, and the voltage was successively ramped from 2110 to 1100 mV over a 3-s period. The patch was excised into ATP-free solution at the arrow.1mM ATP was subsequently added to the intracellular solution as indicated. FIG.2. A, macroscopic currents recorded from four different patches B, mean dose-response relationships for wild-type Kir6.2DC(n 5 11) in response to a series of voltage ramps from 2110 to 1100 mV. Oocytes and for Kir6.2DC-I167M (n 5 5), Kir6.2DC-R50G (n 5 6), and Kir6.2DC- were injected with mRNAs encoding Kir6.2DC, Kir6.2DC-E179Q, E179Q (n 5 7). The slope conductance (G) is given as a fraction of the Kir6.2DC-E167M, or Kir6.2DC-R50G. The patch was excised into ATP- mean conductance (G ) obtained in control solution before and after free solution at the arrows. The dashed line indicates the zero current exposure to ATP. The lines are the best fit of the Hill equation to the level. B, relationship between the increase in conductance on patch exci- data using the mean values for K given in the text. sion and 1/K for channel inhibition by ATP for the channels indicated. The conductance following excision is expressed as a fraction of that in the 3-s voltage ramps from 2110 to 1100 mV, filtered at 0.2 kHz, and cell-attached configuration. The line is drawn to the equation G/G 5 1/(1 sampled at 0.5 kHz. The slope conductance was measured by fitting a 1 ([ATP]/K ) ) with h 5 1.0 and the concentration of ATP 5 5.7 mM. straight line to the data between 220 and 2100 mV; the average of five consecutive ramps was calculated in each solution, except when the tions contained (mM): 140 KCl, 1 K-EGTA, 10 K-HEPES (pH 7.3), with currents were measured immediately after patch excision, when only 2 additions as described. ramps were averaged to avoid errors due to channel rundown. Currents Data Analysis—All data are given as mean 6 S.E. The symbols in the were corrected for leak (,1% of the total current) by subtraction of the figures indicate the mean, and the vertical bars indicate one S.E. (where mean current recorded in Kir6.2DC-injected oocytes at a maximally this is larger than the symbol). ATP dose-response relationships were effective concentration of ATP. fit to the Hill equation: G/G 5 1/(1 1 ([ATP]/K ) )), where G is the slope COSm6 Cell Experiments—Mouse Kir6.2 and hamster SUR1 (Gen- c i TM conductance in the presence of ATP, G is the mean conductance ob- Bank accession number L40623) were used for these experiments. c tained in control solution (before and after ATP application), [ATP] is Point mutations were made in a Kir6.2DC36-C166S construct the ATP concentration, K is the ATP concentration at which inhibition (Kir6.2DCS), in which the C terminus was truncated by 36 amino acids i is half-maximal, and h is the slope factor (Hill coefficient). Statistical and cysteine 166 was replaced by serine. Mutations were prepared by significance was tested using Student’s t test. overlap extension at the junctions of relevant residues using the sequen- tial polymerase chain reaction (16), and the resulting polymerase chain RESULTS reaction products were subcloned into the pCMV6b vector for transfec- Oocyte Experiments—Currents recorded from cell-attached tion. In some experiments, we used a mutant form of SUR1 in which glycine 1485 was replaced by aspartic acid (abbreviated here as patches on oocytes injected with Kir6.2DC were very small but SUR1-GD). increased ;50-fold following patch excision (Fig. 1A). This in- COSm6 cells (a variety of COS cells) were plated at a density of crease in conductance was not observed in water-injected oo- ;2.5 3 10 cells per well in 30-mm 6-well dishes and cultured in cytes, indicating that it results from activation of Kir6.2DC Dulbecco’s modified Eagle’s medium plus 10 mM glucose supplemented currents that are inhibited in the cell-attached configuration. with fetal calf serum (10%), penicillin (100 units/ml), and streptomycin Subsequent application of 1 mM ATP to the intracellular mem- (100 mg/ml). The next day, cells were transfected with pCMV6b- Kir6.2DCS (with mutations as described), pECE-SUR1-GD, and brane surface largely blocked Kir6.2DC currents, suggesting pGreenLantern (Life Technologies, Inc.) using LipofectAMINE reagent that the submembrane ATP concentration may be responsible (Life Technologies, Inc.) according to the manufacturer’s instructions. for channel inhibition in the intact oocyte. Consistent with this Currents were recorded at 250 mV from cell-attached and inside-out idea, large currents were recorded from cell-attached patches patches from green fluorescent cells using a chamber that allowed rapid on oocytes expressing the ATP-insensitive Kir channel Kir1.1a, solution changes (16). Current amplitudes were measured by fitting a and no change in conductance was observed on patch excision. straight line through the data points once the current had reached a steady state. Both bath (intracellular) and pipette (extracellular) solu- The mean Kir1.1a conductance was 15.0 6 3.6 nanosiemens in 30048 A Submembrane ATP Sensor TABLE I Estimation of [ATP] from the increase in conductance following sm patch excision 1/h [ATP] was calculated from [ATP] 5 (G /G 2 1) 3 K , where G is 2 1 i 1 the slope conductance in the cell-attached condition, G is the slope conductance immediately after excision of the same patch, K is the concentration of ATP required to block the channel half-maximally, and h is the Hill coefficient for the dose-response curve for ATP inhibition. Oocytes Clone G G G /G [ATP] n 1 2 2 1 sm nanosiemens mM Kir6.2DC 0.56 6 0.05 28 6251 6 2 5.8 6 0.3 9 Kir6.2DC-E179Q 2.2 6 0.7 41 6922 6 4 5.0 6 0.8 8 Kir6.2DC-I167M 2.6 6 0.8 16.4 6 4.4 7.2 6 0.9 3.2 6 0.5 8 Kir6.2DC-R50G 2.3 6 0.7 5.8 6 1.9 2.4 6 0.2 4.2 6 0.5 4 COSm6 cells Clone G /G [ATP] n 2 1 sm mM Kir6.2DCS-I154C 0.15 6 0.06 1.8 6 0.6 4 Kir6.2DCS 1 SUR1-GD 0.47 6 0.06 0.96 6 0.14 29 Kir6.2DCS-I154C 1 SUR1-GD 0.12 6 0.04 1.4 6 0.3 14 Kir6.2DCS-T171C 1 SUR1-GD 0.65 6 0.10 1.83 6 0.28 19 the cell-attached patch and 15.5 6 3.9 nanosiemens after patch excision (n 5 5, data not shown). The mean conductance in control (water-injected) oocytes was ,0.2 nanosiemens. A critical test of whether the amplitude of Kir6.2DC currents in the intact cell reflects [ATP] is to examine mutant chan- sm nels with altered ATP sensitivity, because these should pro- duce markedly different current amplitudes in cell-attached patches yet yield similar values of [ATP] . We studied three sm different Kir6.2DC mutations that reduce the channel ATP sensitivity to differing extents: R50G, I167M, and E179Q (Fig. 1B). Half-maximal inhibition (K ) of wild-type Kir6.2DC cur- rents was produced by 115 6 6 mM ATP (n 5 11). Mutation of the arginine at position 50 to glycine (R50G) reduced the K for ATP inhibition to 3.4 mM, mutation of isoleucine 167 to methi- onine (I167M) decreased the K to 640 mM, and mutation of FIG.3. Macroscopic currents recorded from four different glutamate 179 to glutamine (E179Q) reduced the K to 300 mM i membrane patches on COSm6 cells transfected with Kir6.2DCS- (17). As expected, channels that showed lower ATP sensitivity I154C, Kir6.2DCS-I154C 1 SUR1-GD, Kir6.2DCS 1 SUR1-GD, or Kir6.2DCS-T171C 1 SUR1-GD. The holding potential was 250 mV. exhibited larger currents in the cell-attached configuration and The patch was excised into ATP-free solution at the arrows. ATP (10 a smaller increase in conductance on patch excision (Fig. 2A mM,1mM, or 0.1 mM) was subsequently added to the intracellular and Table I). There was a reciprocal relationship between the solution as indicated. The dashed line indicates the zero current level. K for current inhibition by ATP and the increase in conduct- ance on patch excision (Fig. 2B), and the [ATP] calculated rents (8, 18) such that coexpressed channels should only be sm from each set of data was similar (see Fig. 4 and Table I). This sensitive to [ATP]. As shown in Fig. 4 and Table I, coexpression constitutes good evidence that the principal cytosolic regulator of SUR1-GD with Kir6.2DCS-I154C does not significantly alter of Kir6.2DC activity is [ATP] . The mean value of the sub- the estimated [ATP] (1.4 6 0.3 mM versus 1.8 6 0.6 mM). sm sm membrane ATP concentration in oocytes calculated from all the We also examined additional Kir6.2DCS mutants that dis- data given in Table I (and Fig. 2) is 4.6 6 0.3 mM (n 5 29) and played a range of ATP sensitivities. For each patch the mem- that from the slope of the graph in Fig. 2B is 5.7 mM. brane current before and immediately after excision was meas- Mammalian Cell Experiments—We also measured the sub- ured, and the ATP dose-response curve was then constructed. membrane ATP concentration in mammalian cells using a sim- The K for ATP inhibition of Kir6.2DCS-I154C 1 SUR1-GD, ilar approach with another C-terminally deleted construct of Kir6.2DCS 1 SUR1-GD, and Kir6.2DCS-T171C 1 SUR1-GD Kir6.2 (Kir6.2DCS-I154C). This contains the double mutation currents were 0.49 6 0.1 (n 5 14), 0.78 6 0.09 (n 5 29), and C166S and I154C, which shifts the ATP sensitivity of the 3.09 6 0.49 mM (n 5 19), respectively. As observed in the oocyte channel into the appropriate physiological range: K 5 0.54 6 experiments, those channels that were least sensitive to ATP 0.12 mM (n 5 4). Table I shows that the estimated [ATP] in exhibited the largest currents in the cell-attached configura- sm COSm6 cells using Kir6.2DCS-I154C was 1.8 1 0.6 mM. The tion and the smallest increment in current on patch excision patch current level in such experiments was generally quite (Fig. 3). The value of [ATP] estimated for the different K sm ATP low. As reported previously (14), the level of current is en- channel mutants varied between 1 and 1.8 mM (Fig. 4 and hanced by coexpression of SUR1. To obtain larger patch cur- Table I), with an overall mean value of 1.36 6 0.13 mM (n 5 66). rents, and hence increase the accuracy of measured channel These values are somewhat lower than those found in oocytes, activities, we performed further experiments on cells coex- suggesting that [ATP] may be lower in COSm6 cells. sm pressing Kir6.2DCS constructs with a mutant SUR1 containing Effects of Metabolic Inhibition—In intact Xenopus oocytes, the point mutation G1485D (SUR1-GD). This mutation abol- inhibition of cell metabolism by 3 mM azide leads to an increase ishes nucleotide diphosphate stimulation of K channel cur- in the whole-cell K current (15). Using the value for resting ATP ATP A Submembrane ATP Sensor 30049 ATP from certain compartments (such as the yolk platelets), or it may indicate an inverse ATP gradient between the mem- brane and bulk cytosol. We favor the former explanation be- cause a similar difference was found in oocytes of the frog Rana pipiens in biochemical measurements of cytosolic (6 mM) and total (2.5 mM) [ATP] (24). Whatever the reason, our results do not support the widely held view that the submembrane ATP concentration is lower than that of the bulk cytosol (1– 4). Recently, the submembrane ATP concentration has been esti- mated in pancreatic b-cells using a membrane-tagged luciferin and found to be ;1mM under resting conditions (3 mM glucose) (25). Our results on mammalian cells are in good agreement with these studies. Furthermore, the fact that two different methods provide a similar value for [ATP] helps validate the different sm assumptions intrinsic to each method. The bulk cytosolic [ATP] FIG.4. Mean calculated ATP concentrations for oocytes (left) in various mammalian cells is also ;1mM: 1.0 mM in b-cells (25), or COSm6 cells (right) expressing the indicated K channel ATP 0.9 mM in HeLa cells (26), and 0.8 –1.2 mM in COS-1 cells (27). constructs in the presence and absence of metabolic inhibitor. The advantage of using Kir6.2DC as a biosensor is that it is able Black bars indicate the ATP concentration in control solution. Gray bars indicate the ATP concentration in the presence of the metabolic to respond very rapidly to changes in [ATP] . sm inhibitor azide (3 mM). In conclusion, our results suggest that Kir6.2DC alone, or Kir6.2 coexpressed with a mutant SUR1, may be used as tools to monitor the submembrane ATP concentration in real time in [ATP] obtained above, we can estimate the fall in submem- sm a single cell. Inside-out patches excised from cells expressing brane [ATP] following metabolic inhibition. Fig. 4 shows the mutant K channels might also be used to detect the local estimated [ATP] in the presence of 3 mM azide. The mean ATP sm release of ATP from purinergic neurons, as described for ace- [ATP] , calculated from all data, fell to 1.2 6 0.1 mM (n 5 24) sm tylcholine release using acetylcholine receptor channels (28). In when metabolism was inhibited. This value is similar to that a similar fashion, inside-out macropatches containing mutant measured biochemically for the bulk cytoplasm in the presence K channels might serve to monitor exocytosis from single of3mM azide (1.7 mM, Ref. 15). ATP secretory cells, as many types of secretory granules contain ATP. Submembrane [ATP] also fell in mammalian cells incubated Importantly, our data also demonstrate that the submembrane in3mM azide (Fig. 4) to a mean value of 0.76 6 0.11 mM (n 5 ATP concentration lies in the millimolar, rather than the micro- 12) after 10 min (averaged for all Kir6.2DCS mutants coex- molar, range in both Xenopus oocytes and mammalian cells. pressed with SUR1-GD). Even greater falls in [ATP] were sm observed when cells were preincubated in the presence of 1 mM REFERENCES 2-deoxyglucose and 2.5 mg/ml oligomycin (Fig. 4); in this case, the 1. Ashcroft, F. M., and Ashcroft, S. J. H. (1990) Cell. Signalling 2, 197–214 2. Niki, I., Ashcroft, F. M., and Ashcroft, S. J. H. (1989) FEBS Lett. 257, 361–364 mean value of [ATP] was 0.1 6 0.06 mM (n 5 5) after a 30-min sm 3. Aw, T. Y., and Jones, D. P. (1985) Am. J. Physiol. 249, C385–C392 preincubation (measured for Kir6.2DCS mutants coexpressed 4. Jones, D. P. (1985) Am. J. Physiol. 250, C663–C675 with SUR1-GD). 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(1997) J. Gen. Physiol. 110, 655– 664 11. Inagaki, N., Gonoi, T., Clement, J. P., IV, Namba, N., Inazawa, J., Gonzalez, similar estimate of [ATP] is obtained for channels with differ- sm G., Aguilar-Bryan, L., Seino, S., and Bryan, J. (1995) Science 270, ent K for ATP inhibition. We have shown elsewhere that 1166 –1169 12. Sakura, H., Amma ¨la ¨ , C., Smith, P. A., Gribble, F. M., and Ashcroft, F. M. Kir6.2DC is selectively inhibited by ATP and is not substantially (1995) FEBS Lett. 377, 338 –344 blocked by other nucleoside triphosphates (K . 5mM, Ref. 17). 13. Inagaki, N., Gonoi, T., Clement, J. P., IV, Wang, C. Z., Aguilar-Bryan, L., Only ATP, Ap A(K 5;100 mM), and ADP (K 5;250 mM) were Bryan, J., and Seino, S. (1996) Neuron 16, 1011–1017 4 i i 14. Tucker, S. J., Gribble, F. M., Zhao, C., Trapp, S., and Ashcroft, F. M. (1997) potent blockers of Kir6.2DC. The concentrations of Ap A and Nature 387, 179 –183 ADP in the bulk cytoplasm are believed to lie within the low and 15. 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(1991) phatidylinositol bisphosphate from the membrane that occurs J. Biol. Chem. 266, 22887–22892 following patch excision is accompanied by an enhanced ATP 21. Fan, Z., and Makielski, J. C. (1997) J. Biol. Chem. 272, 5388 –5395 22. Shyng, S.-L., and Nichols, C. G. (1998) Science 282, 1138 –1141 sensitivity (21–23). Such a lowering of the K for ATP inhibition 23. Baukrowitz, T., Schulte, U., Oliver, D., Herlitze, S., Krauter, T., Tucker, S. J., would lead to an underestimate of [ATP] . Thus to avoid sm Ruppersberg, J. P., and Fakler, B. (1998) Science 282, 1141–1144 artifactual changes in ATP sensitivity, ATP dose-response 24. Miller, D. S., and Horowitz, S. B. (1986) J. Biol. Chem. 261, 13911–13915 25. Kennedy, H. J., Pouli, A. E., Ainscow, E. K., Jouaville, L. S., Rizzuto, R., and curves were measured immediately after patch excision. Rutter, G. A. (1999) J. Biol. Chem. 274, 13281–13291 The mean value of the submembrane ATP concentration in 26. Wang, R. H, Tao, L., Trumbore, M. W., Berger, S. L. (1997) J. Biol. Chem. 272, 26405–26412 oocytes (;5mM) is approximately double that measured from 27. Sippel, C. J., Dawson, P. A., Shen, T., and Perlmutter, D. H. (1997) J. Biol. the whole oocyte using a biochemical method (2.3 mM, Ref. 15). Chem. 272, 18290 –18297 The difference may be only apparent and reflect exclusion of 28. Hume, R. I., Role, L. W., and Fischbach, G. D. (1983) Nature 305, 632– 634

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Published: Sep 1, 2000

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