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The ATP-sensitive potassium (KATP) channel couples glucose metabolism to insulin secretion in pancreatic β-cells. It comprises regulatory sulfonylurea receptor 1 and pore-forming Kir6.2 subunits. Binding and/or hydrolysis of Mg-nucleotides at the nucleotide-binding domains of sulfonylurea receptor 1 stimulates channel opening and leads to membrane hyperpolarization and inhibition of insulin secretion. We report here the first purification and functional characterization of sulfonylurea receptor 1. We also compared the ATPase activity of sulfonylurea receptor 1 with that of the isolated nucleotide-binding domains (fused to maltose-binding protein to improve solubility). Electron microscopy showed that nucleotide-binding domains purified as ring-like complexes corresponding to ∼ 8 momomers. The ATPase activities expressed as maximal turnover rate [in nmol Pi·s−1·(nmol protein)−1] were 0.03, 0.03, 0.13 and 0.08 for sulfonylurea receptor 1, nucleotide-binding domain 1, nucleotide-binding domain 2 and a mixture of nucleotide-binding domain 1 and nucleotide-binding domain 2, respectively. Corresponding Km values (in mm) were 0.1, 0.6, 0.65 and 0.56, respectively. Thus sulfonylurea receptor 1 has a lower Km than either of the isolated nucleotide-binding domains, and a lower maximal turnover rate than nucleotide-binding domain 2. Similar results were found with GTP, but the Km values were lower. Mutation of the Walker A lysine in nucleotide-binding domain 1 (K719A) or nucleotide-binding domain 2 (K1385M) inhibited the ATPase activity of sulfonylurea receptor 1 by 60% and 80%, respectively. Beryllium fluoride (Ki 16 µm), but not MgADP, inhibited the ATPase activity of sulfonylurea receptor 1. In contrast, both MgADP and beryllium fluoride inhibited the ATPase activity of the nucleotide-binding domains. These data demonstrate that the ATPase activity of sulfonylurea receptor 1 differs from that of the isolated nucleotide-binding domains, suggesting that the transmembrane domains may influence the activity of the protein.
ATP-sensitive potassium (KATP) channels couple cell metabolism to membrane excitability and transmembrane ion fluxes. In pancreatic β-cells, they are of crucial importance for regulating insulin secretion . At substimulatory glucose concentrations, KATP channels are open and generate a negative potential that keeps voltage-gated Ca2+ channels closed and abolishes Ca2+ influx. Because a rise in intracellular Ca2+ is needed to stimulate insulin granule release, this prevents insulin secretion. When plasma glucose levels increase, glucose uptake and metabolism lead to changes in the intracellular concentrations of adenine nucleotides that close KATP channels, triggering Ca2+ channel opening, Ca2+ influx, elevation of intracellular Ca2+ and insulin release.
The β-cell KATP channel is a large octameric complex that comprises a central tetrameric Kir6.2 pore surrounded by four sulfonylurea receptor (SUR) 1 subunits . Both Kir6.2 and SUR1 subunits are involved in the metabolic regulation of channel activity: ATP binding to Kir6.2 causes channel inhibition , whereas interaction of Mg-nucleotides (MgATP and MgADP) with SUR1 stimulates channel opening [4–6]. Impairment of nucleotide interactions with either subunit can lead to neonatal diabetes or its converse, congenital hyperinsulinism .
SUR belongs to the ATP-binding cassette (ABC) protein superfamily . It has 17 transmembrane helices and two large cytosolic loops, which contain the nucleotide-binding domains (NBDs) NBD1 and NBD2. As in all ABC proteins, each NBD contains a highly conserved Walker A (WA) and Walker B (WB) motif involved in ATP binding and hydrolysis, an invariant ‘signature sequence’, and several other conserved residues. Crystallization of a number of prokaryotic NBDs and ABC proteins indicates that they associate in a sandwich dimer conformation [8–11], in which residues from the WA and WB motifs of one NBD interact with the signature sequence of the other NBD to form separate ATP-binding sites, with distinct properties. Each ATP-binding site therefore contains contributions from both NBD1 and NBD2. Evidence of physical interaction between the NBDs, and molecular modeling studies, support the idea that SUR1 also conforms to the sandwich dimer model [12,13]. Functional studies demonstrate that formation of such a sandwich dimer is critical for driving gating of cystic fibrosis transmembrane conductance regulator (CFTR) channels , but this has not yet been demonstrated for KATP channels.
There are two genes that encode SUR, ABCC8 (SUR1) and ABCC9 (SUR2) [15–17]. The latter exists in several splice variants, the most important being SUR2A and SUR2B. Differences in the SUR subunit contribute to the variable metabolic sensitivities of KATP channels in different tissues. For example, even when heterologously expressed in the same cell, recombinant Kir6.2–SUR2A channels open less readily on metabolic inhibition than Kir6.2–SUR1 channels . It has been suggested that this may relate to differences in the ATPase activity of SUR1 and SUR2 .
The ATPase activity of full-length SUR1 has not been measured directly to date. However, MgATP hydrolysis has been measured directly for recombinant proteins in which either NBD1 or NBD2 of SUR was fused to the maltose-binding protein [19–21]. ATPase activity of native SUR (i.e. containing both NBDs and transmembrane domains) has also been inferred by comparing covalent labeling with 8-azido-[32P]ATP[αP] and 8-azido-[32P]ATP[γP]. In these studies, however, hydrolysis by NBD2, but not NBD1, was detected. Unlike prokaryotic ABC proteins, NBD1 and NBD2 of SUR1 show significant sequence differences: thus, the ATPase activity of the isolated recombinant NBD homodimers will not necessarily reflect that of the NBD heterodimer expected for native SUR1. Furthermore, the presence of the transmembrane domains in SUR1 may influence ATPase activity. We have therefore purified SUR1 and compared its capacity to hydrolyze ATP and GTP with that of isolated NBD1, or NBD2, of SUR1 [fused to maltose-binding protein (MBP)]. We also measured the ATPase activity of a mixture of NBD1 and NBD2 proteins. In addition, the effects of the inhibitors beryllium fluoride (BeF) and MgADP were explored.