Transduction of energetic signals into membrane electrical events governs vital cellular functions, ranging from hormone secretion and cytoprotection to appetite control and hair growth. Central to the regulation of such diverse cellular processes are the metabolism sensing ATP-sensitive K ؉ (KATP) channels. However, the mechanism that communicates metabolic signals and integrates cellular energetics with K ATP channel-dependent membrane excitability remains elusive. Here, we identify that the response of KATP channels to metabolic challenge is regulated by adenylate kinase phosphotransfer. Adenylate kinase associates with the KATP channel complex, anchoring cellular phosphotransfer networks and facilitating delivery of mitochondrial signals to the membrane environment. Deletion of the adenylate kinase gene compromised nucleotide exchange at the channel site and impeded communication between mitochondria and K ATP channels, rendering cellular metabolic sensing defective. Assigning a signal processing role to adenylate kinase identifies a phosphorelay mechanism essential for efficient coupling of cellular energetics with K ATP channels and associated functions. D elivery of metabolic signals to intracellular compartments is a critical determinant of cellular homeostasis. In particular, efficient communication between cellular energetics and membrane metabolic sensors is required for regulation of cell excitability and associated functions (1, 2). Plasmalemmal ATPsensitive K ϩ (K ATP ) channels, formed by the Kir6.2 potassium channel and the sulfonylurea receptor (SUR), are unique nucleotide sensors that adjust membrane potential in response to intracellular metabolic oscillations (2-5). Transition of the SUR subunit from the ATP to the ADP-liganded state promotes K ϩ permeation through Kir6.2 and defines K ATP channel activity (5-7). However, the mechanism that facilitates nucleotide exchange in the K ATP channel environment and promotes coupling of membrane electrical events with cellular metabolic pathways remains unknown.Cellular phosphotransfer reactions catalyze nucleotide exchange facilitating communication between sites of ATP generation and ATP utilization (8-11). In this way, the phosphotransfer enzyme adenylate kinase (AK) amplifies metabolic signals and promotes intracellular phosphoryl transfer by catalyzing the reaction ATP ϩ AMP 7 2ADP (12, 13). Adenylate kinase has a distinct signaling role in setting the cellular response to stress through activation of AMP-dependent processes (12-15). Deletion of the major AK isoform (AK1) results in disturbed muscle energetic economy and decreased tolerance to metabolic stress (14, 15). Mutations in AK compromise nucleotide export from mitochondria (16), as well as the function of ATP-binding cassette transporters (17). Conversely, stimulation of AK phosphotransfer promotes nucleotide-dependent membrane functions (18,19). However, the actual significance of AK phosphotransfer in communicating energetic signals to membrane metabolic sensors, such as the K ATP cha...

ATP-sensitive K+ (KATP) channels are unique metabolic sensors formed by association of Kir6.2, an inwardly rectifying K+ channel, and the sulfonylurea receptor SUR, an ATP binding cassette protein. We identified an ATPase activity in immunoprecipitates of cardiac KATP channels and in purified fusion proteins containing nucleotide binding domains NBD1 and NBD2 of the cardiac SUR2A isoform. NBD2 hydrolyzed ATP with a twofold higher rate compared to NBD1. The ATPase required Mg2+ and was insensitive to ouabain, oligomycin, thapsigargin, or levamisole. K1348A and D1469N mutations in NBD2 reduced ATPase activity and produced channels with increased sensitivity to ATP. KATP channel openers, which bind to SUR, promoted ATPase activity in purified sarcolemma. At higher concentrations, openers reduced ATPase activity, possibly through stabilization of MgADP at the channel site. K1348A and D1469N mutations attenuated the effect of openers on KATP channel activity. Opener-induced channel activation was also inhibited by the creatine kinase/creatine phosphate system that removes ADP from the channel complex. Thus, the KATP channel complex functions not only as a K+ conductance, but also as an enzyme regulating nucleotide-dependent channel gating through an intrinsic ATPase activity of the SUR subunit. Modulation of the channel ATPase activity and/or scavenging the product of the ATPase reaction provide novel means to regulate cellular functions associated with KATP channel opening.

Structurally unique among ion channels, ATP-sensitive K؉ (K ATP ) channels are essential in coupling cellular metabolism with membrane excitability, and their activity can be reconstituted by coexpression of an inwardly rectifying K ؉ channel, Kir6. Potassium channels are the most diverse group of ion channels, with molecular cloning revealing a number of structurally distinct families, including the subfamily of inwardly rectifying K ϩ (Kir) channels (11,27,35). Channel diversity is increased by the ability of constitutive subunits to form not only homomeric but also heteromultimeric complexes with distinct functional and regulatory properties (8,9,15,21,27,30,39,53). Present in most excitable tissues, ATP-sensitive K ϩ (K ATP ) channels belong to the Kir family and are involved in signaling networks that transduce cellular metabolic events into membrane potential changes (1, 9, 40). These channels are regulated by intracellular nucleotides and have been implicated in hormone secretion, cardioprotection, and neurotransmitter release, with their function best understood in the pancreatic ␤ cell, where K ATP channels are essential in glucose-mediated membrane depolarization and insulin secretion (7,9,14,31,34,42,44,52). Structurally unique among K ϩ channels, K ATP channel activity can be reconstituted by coexpressing two unrelated proteins: the Kir channel Kir6.2 and the ATP-binding cassette (ABC) protein SUR, specifically the SUR1 isoform for the pancreatic channel phenotype (2, 22, 38). Expression of Kir6.2 alone does not result in functional ion channels, suggesting an intimate and required interaction between Kir6.2 with SUR1 (1, 7, 40, 41). Actually, expression of Kir6.2-SUR1 fusion constructs indicates that a subunit stoichiometry of 1:1 is necessary for assembly of active K ATP channels (10, 24). Furthermore, Kir6.2 and SUR1 genes are clustered on chromosome 11 (p15.1), separated by a short intergenic sequence of 4.3 kb, suggesting that these genes could be cotranscribed and cotranslated to form a functional heteromultimeric channel (1,9,22,40). To date, evidence for physical association between Kir6.2 and SUR1 is based on photoaffinity labeling of both channel subunits by radioactive sulfonylurea (10). Labeling of Kir6.2 was dependent on coexpression of SUR1, suggesting close association between the two subunits (10). However, photoaffinity labeling is based primarily on proximity rather than physical interaction between proteins (18).Recent evidence indicates that K ϩ channels are tetramers of single subunits comprising the K ϩ -selective pore (27). The measurement of K ATP channel activity in cells expressing mutant carboxy-truncated Kir6.2 has been interpreted to mean that the presence of the carboxy terminus in Kir6.2 prevents functional expression of the channel in the absence of SUR (51). However, it is not known whether the distal carboxy terminus of Kir6.2 merely serves as a suppressor of channel * Corresponding author. Mailing address: Guggenheim 7, Mayo Clinic, Rochester, MN 55905.

No abstract

The ATP-sensitive potassium (KATP) channel is thought to play an important role in the protection of heart and brain against tissue hypoxia. The genetic regulation of the components of the channel by hypoxia has not been previously described. Here, we investigated the regulation of the two pore-forming channel proteins, Kir6.1 and Kir6.2, in response to hypoxia in vivo and in vitro. We find that these two structurally-related inwardly-rectifying potassium channel proteins are reciprocally regulated by hypoxia in vivo, with upregulation of Kir6.1 and down-regulation of Kir6.2, thereby resulting in a significant change in the composition of the channel complex in response to hypoxia. In vitro we describe neuronal and cardiac cell lines in which Kir6.1 is up-regulated by hypoxia, demonstrating that Kir6.1 is a hypoxia-inducible gene. We conclude that the heart and brain display genetic plasticity in response to hypoxic stress through specific genetic reprograming of cytoprotective channel genes.