期刊名称：Proceedings of the National Academy of Sciences
印刷版ISSN：0027-8424
电子版ISSN：1091-6490
出版年度：2004
卷号：101
期号：1
页码：76-81
DOI：10.1073/pnas.0306347101
语种：English
出版社：The National Academy of Sciences of the United States of America
摘要：ATP-sensitive K+ (KATP) channels play important roles in the regulation of membrane excitability in many cell types. ATP inhibits channel activity by binding to a specific site formed by the N and C termini of the pore-forming subunit, Kir6.2, but the structural changes associated with this interaction remain unclear. Here, we use fluorescence resonance energy transfer (FRET) to study the ATP-dependent interaction between the N and C termini of Kir6.2 using a construct bearing fused cyan and yellow fluorescent proteins (ECFP-Kir6.2-EYFP). When expressed in human embryonic kidney cells, ECFP-Kir6.2-EYFP/SUR1 channels displayed FRET that was augmented by agonist stimulation and diminished by metabolic poisoning. Addition of ATP to permeabilized cells or isolated plasma membrane sheets increased FRET. FRET changes were abolished by Kir6.2 mutations that altered ATP-dependent channel closure and channel gating. In the wild-type channel, the ATP concentrations, which increased FRET (EC50 = 1.36 mM), were significantly higher than those causing channel inhibition (IC50 = 0.29 mM). Demonstrating the existence of intermolecular interactions, a dimeric construct comprising two molecules of Kir6.2 linked head-to-tail (ECFP-Kir6.2-Kir6.2-EYFP) displayed less FRET than the monomer in the absence of nucleotide but still exhibited ATP-dependent FRET increases (EC50 = 1.52 mM) and channel inhibition. We conclude that binding of ATP to Kir6.2, (i) alters the interaction between the N- and C-terminal domains, (ii) probably involves both intrasubunit and intersubunit interactions, (iii) reflects ligand binding not channel gating, and (iv) occurs in intact cells when subplasmalemmal [ATP] changes in the millimolar range.
