K _ATP channel inhibition by ATP requires distinct functional domains of the cytoplasmic C terminus of the pore-forming subunit

Abstract

ATP-sensitive potassium (''K ATP '') channels are rapidly inhibited by intracellular ATP. This inhibition plays a crucial role in the coupling of electrical activity to energy metabolism in a variety of cells. The K ATP channel is formed from four each of a sulfonylurea receptor (SUR) regulatory subunit and an inwardly rectifying potassium (K ir 6.2) pore-forming subunit. We used systematic chimeric and point mutagenesis, combined with patch-clamp recording, to investigate the molecular basis of ATP-dependent inhibition gating of mouse pancreatic ␤ cell K ATP channels expressed in Xenopus oocytes. We identified distinct functional domains of the presumed cytoplasmic C-terminal segment of the K ir 6.2 subunit that play an important role in this inhibition. Our results suggest that one domain is associated with inhibitory ATP binding and another with gate closure.The ATP-sensitive potassium (K ATP ) channel couples membrane electrical activity to energy metabolism in a variety of cells and is important in several physiological systems. In pancreatic ␤ cells, for example, it is essential for coupling the rate of insulin release to blood glucose levels. Although the channel's name reflects its characteristic inhibition by intracellular ATP, little is known about the molecular nature of this property.To understand the molecular mechanisms underlying ATPdependent inhibition gating in the K ATP channel, one must identify those parts of the channel complex that form the ATP-binding site, the inhibition gate, and the ''linkage'' domains handling signal flow between them. The K ATP channel is assembled from four each of two subunit types, a regulatory sulfonylurea receptor (SUR) or SUR1 and a potassium poreforming subunit or K ir 6.2 (1-4). SUR1 is a member of the ATP-binding cassette (ABC) family of proteins featuring two cytoplasmic nucleotide-binding folds, viewed initially as likely mediators of inhibition by ATP (5, 6). However, a mutant K ir 6.2 in which the C-terminal 26 residues are deleted remarkably gives rise to potassium channels that retain much ATP sensitivity in the absence of SUR1 (7). The truncation does, however, diminish ATP sensitivity of the K ATP channel 10-fold, as does SUR2 when coexpressed with wild-type K ir 6.2 (6). These results have led to opposing models in which ATP acts either on K ir 6.2 or on SUR to inhibit the K ATP channel.We tested whether major components of the ATPdependent inhibition gating mechanism reside in the poreforming subunit. By systematically mutating K ir 6.2, we localized molecular components of ATP-dependent inhibition gating to distinct regions of its cytoplasmic C-terminal segment. Our results confirm and extend the findings of Tucker et al. (7) that the primary site of action of inhibitory ATP lies on K ir 6.2. We go on to show that one of the regions we identified is likely associated with inhibitory ATP binding, whereas a second region appears to be associated with inhibition gate closure. MATERIALS AND METHODSMolecular Biology. Cloning of mouse SUR1 and K ...

Nucleotide-gated KATPchannels integrated with creatine and adenylate kinases: Amplification, tuning and sensing of energetic signals in the compartmentalized cellular environment

“…Nucleotide-dependent K ATP channel gating was simulated by an allosteric model where: (i) 4 identical binding sites for ATP and ADP co-exist within the octameric stoichiometry of the K ATP channel complex [15,19]; (ii) binding of ATP to the pore-forming Kir6.2 subunit inhibits channel opening [16,17]; (iii) binding of ADP to the regulatory SUR subunit antagonizes ATPbinding to Kir6.2 [18,20,21]. T i and D i (i = 0-4) are channel species with bound ATP and/or ADP.…”

“…1) [13][14][15]. ATP maintains K ATP channel closure by binding to Kir6.2 [16,17], whereas ATP/ADP interactions with SUR secure the metabolic sensor function of the channel complex (Fig. 1A) [18][19][20].…”

Neuroprotective role of ATP-sensitive potassium channels in cerebral ischemia

“…The fast gating kinetics of the channels is determined by an intrinsic structure within the pore-loop that is near the selectivity filter of the Kir6.2 subunit [41,44] . The long last interburst closing kinetics requires ATP binding to the TM2 helices [42,45] .…”

“…ATP binds to the cytoplasmic domain of the Kir6.2 subunit [44,47,48] to inhibit the channel [49] . Each Kir6.2 subunit provides one ATP binding pocket, which is constituted by residues R50, I182, K185, R201, and G334 [5,36] .…”

ABCC8 and ABCC9: ABC transporters that regulate K+ channels

“…Multiple studies have shown that engineered K IR 6.x subunits can assemble functional pores in the absence of a SUR, and that their activity is sensitive to ATP [8, 43,150]. Biochemical studies have demonstrated ATP binding to the large C-terminal domain [147,148,151].…”