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

ATP-sensitive potassium channels are under complex regulation by intracellular ATP and ADP. The potentiating effect of MgADP is conferred by the sulfonylurea receptor subunit of the channel, SUR, whereas the inhibitory effect of ATP appears to be mediated via the pore-forming subunit, Kir6 ATP-sensitive potassium (K ATP )1 channels play important roles in many tissues by linking the metabolic status of the cell to its membrane potential (1, 2). In pancreatic ␤-cells, K ATP channels are critical for the regulation of glucose-induced insulin secretion (3, 4) and have recently been shown to be an octameric complex of two subunits, which coassemble with a 4:4 stoichiometry (5-9). The pore-forming subunit, Kir6.2, is a member of the inwardly rectifying K ϩ channel family (10, 11), whereas the other subunit, the sulfonylurea receptor (SUR1), is a member of the ATP-binding cassette transporter superfamily (12, 13). Unlike most other Kir channels, expression of Kir6.2 alone does not produce functional channel activity; instead, it requires coexpression with SUR1. However, an isoform of Kir6.2 in which the last 26 amino acids have been removed (Kir6.2⌬C26) is capable of expressing functional K ϩ channel activity in the absence of SUR1. Kir6.2⌬C26 retains sensitivity to inhibition by ATP, and mutations in this subunit can significantly reduce the inhibitory effect of ATP (14,15). This has been taken as evidence that the primary site at which ATP acts to cause K ATP channel closure resides on Kir6.2. However, controversy still remains as to whether ATP binds directly to Kir6.2, whether truncation of Kir6.2 exposes a cryptic blocking site for nucleotides, or whether ATP inhibition is mediated indirectly by binding of the nucleotide to an endogenous subunit that modulates the activity of Kir6.2 (9,14). In the present study, we show that Kir6.2 directly binds the photoaffinity analog of ATP, 8-azido-ATP, and that this labeling can be reduced by 50% with 100 M ATP. We also demonstrate that the related inwardly rectifying K ϩ channel subunit Kir4.1, which is not inhibited by ATP, exhibits no significant photoaffinity labeling by 8-azido-[␥-32 P]ATP. Furthermore, we show that mutations in Kir6.2 that reduce the inhibitory effect of ATP on channel activity also reduce photoaffinity labeling. This provides strong evidence that ATP binds directly to Kir6.2. MATERIALS AND METHODSTransfection and Preparation of Membranes-COS-7 cells were cultured in Dulbecco's modified Eagle's medium with 10% fetal bovine serum in a humidified atmosphere of 95% air, 5% CO 2 at 37°C. COS-7 cells were transfected with expression vectors encoding full-length wildtype or mutant mouse Kir6.2, tagged with the Flag epitope at the NH 2 terminus and with a hexahistidine tag at the COOH terminus (FlagKir6.2), or tagged with the Flag epitope at the COOH terminus (Kir6.2-Flag), using LipofectAMINEplus (Life Technologies, Inc.) according to the manufacturer's directions. Rat Kir4.1 was tagged with the Flag epitope at the COOH terminus (Kir4

The 70-kDa peroxisomal membrane protein (PMP70) and adrenoleukodystrophy protein (ALDP), half-size ATP-binding cassette transporters, are involved in metabolic transport of long and very long chain fatty acids into peroxisomes. We examined the interaction of peroxisomal ATP-binding cassette transporters with ATP using rat liver peroxisomes. PMP70 was photoaffinitylabeled at similar efficiencies with 8-azido-[␣-32 P]ATP and 8-azido-[␥-32 P]ATP when peroxisomes were incubated with these nucleotides at 37°C in the absence Mg 2؉ and exposed to UV light without removing unbound nucleotides. The photoaffinity-labeled PMP70 and ALDP were co-immunoprecipitated together with other peroxisomal proteins, which also showed tight ATP binding properties. Addition of Mg 2؉ reduced the photoaffinity labeling of PMP70 with 8-azido-[␥-32 P]ATP by 70%, whereas it reduced photoaffinity labeling with 8-azido-[␣-32 P]ATP by only 20%. However, two-thirds of nucleotide (probably ADP) was dissociated during removal of unbound nucleotides. These results suggest that ATP binds to PMP70 tightly in the absence of Mg 2؉ , the bound ATP is hydrolyzed to ADP in the presence of Mg 2؉ , and the produced ADP is dissociated from PMP70, which allows ATP hydrolysis turnover. Properties of photoaffinity labeling of ALDP were essentially similar to those of PMP70. Vanadate-induced nucleotide trapping in PMP70 and ALDP was not observed. PMP70 and ALDP were also phosphorylated at a tyrosine residue(s). ATP binding/hydrolysis by and phosphorylation of PMP70 and ALDP are involved in the regulation of fatty acid transport into peroxisomes.ATP-binding cassette (ABC) 1 superfamily proteins are composed of two homologous halves, each of which typically contains six transmembrane ␣ helices and a nucleotide binding fold (NBF). Prominent members of eukaryotic ABC superfamily proteins, such as the multidrug efflux pump MDR1 (ABCB1) and the cystic fibrosis transmembrane conductance regulator CFTR (ABCC7), are full size and contain 12 transmembrane ␣ helices and 2 NBFs. On the other hand, most of the organelle ABC superfamily proteins, such as antigen transporters TAP1 (ABCB1) and TAP2 (ABCB2) on endoplasmic reticulum membranes and peroxisomal ABC proteins, are half size and contain six transmembrane ␣ helices and one NBF.To date, four peroxisomal ABC proteins have been identified in mammalian peroxisomes: the 70-kDa peroxisomal membrane protein (PMP70, ABCD3), adrenoleukodystrophy protein (ALDP, ABCD1), ALDP-related protein (ALDRP, ABCD2), and PMP70-related protein (P70R, ABCD4) (1-7). These half-size ABC proteins are supposed to work after dimerization. Disruption of the half-size ABC protein gene of Saccharomyces cerevisiae PAT1 (Pxa1) or PAT2 (Pxa2), whose products were identified on peroxisomes, resulted in impaired growth in oleic acid medium, suggesting that Pat1p and Pat2p function as heterodimers (8 -11). Indeed, Liu et al. (12) have shown that homoas well as heterodimerization occurred among the ALDP, AL-DRP, and PMP70 by using the yeast two-hybrid syste...

ATP-binding cassette (ABC) superfamily proteins have divergent functions and can be classified as transporters, channels, and receptors, although their predicted secondary structures are very much alike. Prominent members include the sulfonylurea receptor (SUR1) and the multidrug transporter (MDR1). SUR1 is a subunit of the pancreatic beta-cell K(ATP) channel and plays a key role in the regulation of glucose-induced insulin secretion. SUR1 binds ATP at NBF1, and ADP at NBF2 and the two NBFs work cooperatively. The pore-forming subunit of the pancreatic beta-cell K(ATP) channel, Kir6.2, is a member of the inwardly rectifying K(+) channel family, and also binds ATP. In this article, we present a model in which the activity of the K(ATP) channel is determined by the balance of the action of ADP, which activates the channel through SUR1, and the action of ATP, which stabilizes the long closed state by binding to Kir6.2. The concentration of ATP could also affect the channel activity through binding to NBF1 of SUR1. MDR1, on the other hand, is an ATP-dependent efflux pump which extrudes cytotoxic drugs from cells before they can reach their intracellular targets, and in this way confers multidrug resistance to cancer cells. Both NBFs of MDR1 can hydrolyze nucleotides, and their ATPase activity is necessary for drug transport. The interaction of SUR1 with nucleotides is quite different from that of MDR1. Variations in the interactions with nucleotides of ABC proteins may account for the differences in their functions.

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