Direct Photoaffinity Labeling of Kir6.2 by [γ-32P]ATP-[γ]4-Azidoanilide

Tanabe, Kouichi; Tucker, Stephen J.; Ashcroft, Frances M.; Proks, Peter; Kioka, Noriyuki; Amachi, Teruo; Ueda, Kazumitsu

doi:10.1006/bbrc.2000.2780

Cited by 36 publications

(32 citation statements)

References 19 publications

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“…The essential idea, which resolves the longstanding paradox of how K ATP channels with an IC 50(ATP) ϳ100-fold lower than the estimated cytosolic concentration of ATP can couple membrane excitation with metabolism, is that nucleotides, acting through separate binding sites on K IR s and SURs, exert both Mg-independent inhibitory and Mg-dependent stimulatory actions, respectively. The demonstration that C-terminally truncated K IR 6.2 was inhibited by ATP in the absence of SUR provided the first evidence for an inhibitory nucleotide binding site on K IR 6.2 (7), a result consistent with the low affinity photolabeling of K IR 6.2 with azido-analogs of ATP (35,51). Our data with ATP and other nucleotides argue this noncanonical (see also Ref.…”

Section: Discussionmentioning

confidence: 66%

“…We observed that 8-azido-ATP, with its bulky substitution on the adenine ring, was ϳ10-fold less effective than ATP as an inhibitor of K IR 6.2/ SUR1 channels, although it has a similar affinity for SUR (32)(33)(34). ATP-␥AA is poorly recognized by SUR, but inhibits homomeric K IR 6.2 channels as effectively as ATP (35). We compared the dose-response curves of these ATP analogs, and TNP-ATP, from spontaneously active K IR 6.1-1-1-1/SUR1 4 and K IR 6.2-2-2-2/SUR1 4 channels (Fig.…”

Section: Methodsmentioning

confidence: 91%

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A Conserved Inhibitory and Differential Stimulatory Action of Nucleotides on KIR6.0/SUR Complexes Is Essential for Excitation-Metabolism Coupling by KATP Channels

Babenko

Bryan

2001

Journal of Biological Chemistry

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The mechanism by which ubiquitous adenine nucleotide-gated K IR 6.0 4 /SUR 4 channels link membrane excitability with cellular metabolism is controversial. Is a decreased sensitivity to inhibitory ATP required, or is the Mg-ADP/ATP-dependent stimulatory action of the ATPase, sulfonylurea receptor (SUR), on K IR sufficient to elicit a physiologically significant open channel probability? To evaluate the roles of nucleotide inhibition versus stimulation, we compared K IR 6.1-based K NDP channels with K IR 6.2-based K ATP channels and all possible K IR 6.1/6.2 hybrids. Although K NDP channels are thought to be poorly sensitive to inhibitory ATP and to require Mg-nucleotide diphosphates for activity, we demonstrate that, like K ATP , and hybrid channels, they are inhibited with an IC 50(ATP) 100-fold lower than The primary signaling mechanism that attunes the P O to the cellular metabolic status remains controversial. Observations that K ATP channels burst spontaneously in inside-out patches and show reversible inhibition by submillimolar ATP (4 -6) suggested that opening these K IR s in vivo, in the face of millimolar nucleotide, would necessitate a significant decrease in their sensitivity to ATP. The reduction in the ATP sensitivity of isolated K ATP channels upon treatment with numerous substances has been interpreted as evidence for the plasticity of ATP-inhibitory gating in vivo. Following the report (7) that ATP weakly inhibits K IR 6.2-based channels in the absence of SUR (IC 50(ATP) ϭ ϳ0.2 mM versus ϳ5-20 M with SURs; Refs. 8 and 9), K IR -based plasticity of ATP inhibition has been considered a possible cause of the variable activity observed for K ATP channel isoforms in situ.An alternative idea is that SURs, obligatory regulatory subunits of K ATP channels and members of the ABC superfamily (10, 11), act as nucleotide sensors and, in vivo, SURs would exert a Mg-ADP/ATP-dependent stimulatory action sufficient to overcome the saturated inhibitory effect of nucleotides at noncanonical nucleotide binding site(s) on the K IR (1). This hypothesis is consistent with only a small fraction, ϳ1%, of the maximal NP O for K ATP channel population participating in the regulation of insulin secretion from pancreatic ␤-cells (12) or shortening of action potentials of metabolically inhibited ventricular cardiomyocytes (13,14). The hypothesis is consistent with the marginal effect of a decreased submembrane concentration of ATP on K ATP channel activity in situ, e.g. the enhancement of K ATP currents observed when adenylyl cyclases are maximally stimulated, thus reducing ATP without producing stimulatory MgADP, in cardiomyocytes dialyzed with submillimolar ATP, but not in the perforated patch or conventional whole-cell configuration with millimolar [ATP] i (15). The physiologic significance of the Mg-nucleotide-dependent stimulation of K IR 6.2 has been established by the identification of SUR1 mutations that compromise the stimulatory, but not the inhibitory action of Mg-nucleotides on pancreatic ␤-cell K ATP channels (...

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Section: Discussionmentioning

confidence: 66%

Section: Methodsmentioning

confidence: 91%

A Conserved Inhibitory and Differential Stimulatory Action of Nucleotides on KIR6.0/SUR Complexes Is Essential for Excitation-Metabolism Coupling by KATP Channels

Babenko

Bryan

2001

Journal of Biological Chemistry

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“…Therefore, SUR1 would not stay long in the active state, when the cellular ADP concentration is low. Recently, we demonstrated that ATP binds directly to Kir6.2 (27,28), and the binding is speculated to be involved in stabilization of the long closed state of K ATP channel (33). With the rise in the cellular ATP concentration, Kir6.2 would stay longer in ATP-binding form.…”

Section: Discussionmentioning

confidence: 99%

Different Binding Properties and Affinities for ATP and ADP among Sulfonylurea Receptor Subtypes, SUR1, SUR2A, and SUR2B

Matsuo

Tanabe

Kioka

et al. 2000

Journal of Biological Chemistry

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134

143

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ATP-sensitive potassium (K ATP )1 channels are inwardly rectifying potassium channels, which are inhibited by ATP and stimulated by MgADP (1-3). They play important roles by linking cellular metabolic level to membrane potential by sensing intracellular ATP and ADP levels in various tissues such as pancreatic ␤-cells, heart, brain, skeletal muscle, and vascular smooth muscle. The K ATP channel is a hetero-octamer composed of sulfonylurea receptor (SURx) and Kir6.x subunits in 4:4 stoichiometry (4 -7). SURx is a member of the ATP-binding cassette (ABC) superfamily including P-glycoprotein (MDR1), multidrug resistance-associated protein (MRP1), and the cystic fibrosis transmembrane conductance regulator (CFTR) (8, 9), all of which have two nucleotide-binding folds (NBFs) per molecule; Kir6.x is a member of the inwardly rectifying potassium channel family (10 -12). Both SURx and Kir6.x have a number of subtypes as follows: SUR1, SUR2A, and SUR2B and Kir6.1 and Kir6.2. SUR1 has been cloned as a high affinity binding protein for sulfonylurea (9), the most commonly used drug for treatment of patients with type 2 diabetes. SUR2A shares 68% amino acid identity with SUR1, and SUR2B is a splicing variant of SUR2A differing only in its C-terminal 42 amino acids (13,14). The Cterminal 42 amino acids of SUR2B are similar to those of SUR1. Kir6.1 and Kir6.2 share 71% amino acid identity with each other, both of which have two putative transmembrane domains and an ion pore-forming (H5) region.Pancreatic ␤-cell K ATP channels, composed of SUR1 and Kir6.2, regulate insulin secretion by altering the ␤-cell membrane potential (1,3,15,16). Coexpression of SUR2A/Kir6.2, SUR2B/Kir6.2, and SUR2B/Kir6.1 has been reported to reconstitute cardiac, smooth muscle, and vascular smooth muscle K ATP channels, respectively (2, 3). These channels have different sensitivities to ATP and show different responses to sulfonylurea drugs and potassium channel openers (10,11,14,17,18). The IC 50 (ATP) of SUR1/Kir6.2 K ATP channels is about 10 M, whereas those of SUR2A/Kir6.2 and SUR2B/Kir6.2 are about 100 and 300 M, respectively (10,11,14). SUR2B/Kir6.1 K ATP channels are not inhibited by ATP but are stimulated by ADP and ATP (17). SUR1/Kir6.2 K ATP channels are inhibited by glibenclamide at K i ϳ10 nM, and SUR2A/Kir6.2, SUR2B/ Kir6.2, and SUR2B/Kir6.1 K ATP channels are inhibited with K i values in the low micromolar range (10,11,14,17). Both SUR1/Kir6.2 and SUR2B/Kir6.2 K ATP channels are stimulated by diazoxide, but SUR2A/Kir6.2 K ATP channels are not (10,11,14). The differences between these channels may be caused, at least in part, by differences in SUR subtype. However, it is not clear how SUR subtypes cause the different properties of K ATP channel subtypes.We have already shown that NBF1 of SUR1 is a Mg 2ϩ -independent high affinity ATP-binding site, that NBF2 is a Mg 2ϩ -dependent low affinity ATP-binding site, and that MgADP binding at NBF2 stabilizes the 8-azido-ATP binding at

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“…The discovery that Kir6.2⌬C channels are inhibited by ATP, for example, revealed that ATP (and ADP) interact with Kir6.2 to cause channel closure (18,22). Site-directed mutagenesis has identified a number of residues in Kir6.2 that, when mutated, reduce the channel ATP sensitivity (22)(23)(24), and photoaffinity labeling with radiolabeled ATP analogs confirmed that Kir6.2 indeed binds ATP (25,26). Nucleotide binding does not require Mg 2ϩ , is strongly selective for the adenine base, and both ATP and ADP can mediate channel inhibition (22,27).…”

Section: Properties Of the Nucleotide-binding Sites On Kir62 And Sur1mentioning

confidence: 99%

Metabolic Regulation of the Pancreatic Beta-Cell ATP-Sensitive K+ Channel

2004

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Closure of ATP-sensitive K؉ channels (K ATP channels) is a key step in glucose-stimulated insulin secretion. The precise mechanism(s) by which glucose metabolism regulates K ATP channel activity, however, remains controversial. It is widely believed that the principal determinants are the intracellular concentrations of the metabolic ligands, ATP and ADP, which have opposing actions on K ATP channels, with ATP closing and MgADP opening the channel. However, the sensitivity of the channel to these nucleotides in the intact cell, and their relative contribution to the regulation of channel activity, remains unclear. The precise role of phosphoinositides and long-chain acyl-CoA esters, which are capable of modulating the channel ATP sensitivity, is also uncertain. Furthermore, it is still a matter of debate whether it is changes in the concentration of ATP, of MgADP, or of other agents, which couples glucose metabolism to K ATP channel activity. In this article, we review current knowledge of the metabolic regulation of the K ATP channel and provide evidence that MgADP (or MgATP hydrolysis), acting at the regulatory subunit of the channel, shifts the ATP concentration-response curve into a range in which the channel pore can respond to dynamic changes in cytosolic ATP. This metabolic pas de deux orchestrates the pivotal role of ATP in metabolic regulation of the K ATP channel. Diabetes 53 (Suppl. 3): S113-S122, 2004

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Direct Photoaffinity Labeling of Kir6.2 by [γ-32P]ATP-[γ]4-Azidoanilide

Cited by 36 publications

References 19 publications

A Conserved Inhibitory and Differential Stimulatory Action of Nucleotides on KIR6.0/SUR Complexes Is Essential for Excitation-Metabolism Coupling by KATP Channels

A Conserved Inhibitory and Differential Stimulatory Action of Nucleotides on KIR6.0/SUR Complexes Is Essential for Excitation-Metabolism Coupling by KATP Channels

Different Binding Properties and Affinities for ATP and ADP among Sulfonylurea Receptor Subtypes, SUR1, SUR2A, and SUR2B

Metabolic Regulation of the Pancreatic Beta-Cell ATP-Sensitive K+ Channel

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