The Carboxyl Termini of KATP Channels Bind Nucleotides

Abstract

Glutaraldehyde cross-linking demonstrated the multimerization potential of these COOH termini, suggesting that these cytosolic segments may directly interact in intact tetrameric channels. Thus, the COOH termini of K ATP tetrameric channels contain the nucleotide-binding pockets of these metabolically regulated channels with four potential nucleotide-binding sites/channel tetramer. ATP-sensitive or ATP-regulated potassium (K ATP )1 channels couple metabolism to either cell excitability (Kir6.x) (1-6) or potassium secretion (Kir1.1 in kidney) (7,8) and provide therapeutic targets for diseases including tissue ischemia, diabetes, hypertension, and disorders of potassium homeostasis. K ATP channels are formed by an octameric complex of four pore-forming subunits (Kir6.x or Kir1.1) and four sulfonylurea receptors, SUR1 or SUR2 for Kir6.x (2) or the cystic fibrosis transmembrane conductance regulator or SUR2b for Kir1.1 (9, 10).Although the SUR/fibrosis transmembrane conductance regulator subunits contain nucleotide-binding folds (11, 12), this subunit is not required for ATP-mediated inhibition of K ϩ channel activity. For example, deletion of the last 36 amino acids from the COOH terminus of Kir6.2 (Kir6.2⌬C36) produces functional K ϩ channels in the absence of coexpressed SURs that are sensitive to ATP (13). Nevertheless, SUR subunits are required for ADP-mediated activation of K ATP channels (14 -16). Thus, ATP inhibition of K ATP channel activity is thought to involve direct interaction with Kir subunits despite the lack of identifiable nucleotide-binding motifs. The recent demonstration of the photoaffinity labeling of Kir6.2 channel by 8-azido-[␥-32 P]ATP (17, 18) also supports the direct binding of ATP with the pore-forming subunit of K ATP channels. In addition, mutations in both the NH 2 -and COOH-terminal regions of the Kir6.2 (13, 19 -23) and Kir1.1 (24) subunits alter the EC 50 for ATP-mediated channel gating. Because ATP-mediated inhibition of channel activity must be a complex process involving residues that form an ATP-binding pocket and others that may be required for linking ATP binding to channel closure, those mutational studies of channel gating by nucleotides do not provide unequivocal evidence for direct involvement of those residues in ATP binding.In the present study, we assessed the direct binding of fluorescent 2Ј,3Ј-O-(2,4,6-trinitrophenylcyclo-hexadienylidene) adenosine triphosphate (TNP-ATP) to purified maltose-binding fusion proteins of the cytosolic NH 2 and COOH termini of the three known K ATP channels and the COOH terminus of a ATP-insensitive inward rectifier K ϩ channel, Kir2.1 (25). We provide herein what we believe to be the first evidence of direct binding of ATP to cytosolic domains of the pore-forming subunits of K ATP channels and show that the COOH termini, but not the NH 2 termini, of Kir subunits of K ATP channels bind TNP-ATP. The kinetic analyses of TNP-ATP binding suggest that the COOH termini have a single nucleotide-binding site. Based on glutaraldehyde cross-linking...

Localization of the ATP/Phosphatidylinositol 4,5 Diphosphate-binding Site to a 39-Amino Acid Region of the Carboxyl Terminus of the ATP-regulated K+ Channel Kir1.1

“…TNP-ATP Binding-Binding of ATP to these recombinant fusion proteins was performed generally as described previously (19). Briefly, 5 M recombinant protein was dissolved in 50 mM Tris-HCl at pH 7.5, and TNP-ATP binding was detected by the increase in fluorescence (Ex ϭ 403 nm; 546 nm; slit widths 5 nm) upon binding to recombinant protein using a Fluromax-3 spectrofluorometer (Jobin Yvon Inc., Edison, NJ).…”

“…Production and Purification of Maltose-binding Fusion ProteinsRecombinant proteins were expressed as previously described by us (19). Briefly, 1 liter of Luria-Bertani medium with 0.1 mg/ml ampicillin and 0.2% glucose was inoculated with 10 ml of an overnight culture of BL21 cells expressing the fusion vector and grown to an A 600 of ϳ0.5 at 37°C.…”

“…We recently localized the ATP-binding region to the COOH terminus in each of the three ATP-regulated channels: Kir1.1, Kir6.1, and Kir6.2 (19). We found that maltose-binding (MBP) fusion proteins containing Kir1.1 or Kir6.x COOH termini are efficiently produced in bacteria, are soluble without detergent, and directly bind TNP-ATP with a stoichiometry of at least one ATP per COOH-terminal monomer.…”

“…Because these arginine residues are conserved in the Kir6.x channels, similar domains in these channels are likely to also form ATP-binding pockets. (19,24). The deletions and single amino acid substitutions in the COOH terminus of Kir1.1 were generated using PCR.…”

ABCC8 and ABCC9: ABC transporters that regulate K+ channels

“…Biochemical studies have demonstrated ATP binding to the large C-terminal domain [147,148,151]. Recent studies have used the coordinates of bacterial K + channels [42,82,110] to make homology models the K IR 6.x pore and provide novel information on the adenine-selective, nucleotide-diphosphate-binding pocket, which is composed of residues from both the N-and C-termini (7, 29; reviewed in 5).…”