Michael V. Mikhailov

3-D structural and functional characterization of the purified KATP channel complex Kir6.2-SUR1.

ATP‐sensitive potassium (KATP) channels conduct potassium ions across cell membranes and thereby couple cellular energy metabolism to membrane electrical activity. Here, we report the heterologous expression and purification of a functionally active KATP channel complex composed of pore‐forming Kir6.2 and regulatory SUR1 subunits, and determination of its structure at 18 Å resolution by single‐particle electron microscopy. The purified channel shows ATP‐ase activity similar to that of ATP‐binding cassette proteins related to SUR1, and supports Rb+ fluxes when reconstituted into liposomes. It has a compact structure, with four SUR1 subunits embracing a central Kir6.2 tetramer in both transmembrane and cytosolic domains. A cleft between adjacent SUR1s provides a route by which ATP may access its binding site on Kir6.2. The nucleotide‐binding domains of adjacent SUR1 appear to interact, and form a large docking platform for cytosolic proteins. The structure, in combination with molecular modelling, suggests how SUR1 interacts with Kir6.2.

Molecular structure of the glibenclamide binding site of the β-cell KATP channel

We have investigated the structure of the glibenclamide binding site of pancreatic β‐cell ATP‐sensitive potassium (KATP) channels. KATP channels are a complex of four pore‐forming Kir6.2 subunits and four sulfonylurea receptor (SUR1) subunits. SUR1 (ABCC8) belongs to the ATP binding cassette family of proteins and has two nucleotide binding domains (NBD1 and NBD2) and 17 putative transmembrane (TM) sequences. Co‐expression in a baculovirus expression system of two parts of SUR1 between NBD1 and TM12 leads to restoration of glibenclamide binding activity, whereas expression of either individual N‐ or C‐terminal part alone gave no glibenclamide binding activity, confirming a bivalent structure of the glibenclamide binding site. By using N‐terminally truncated recombinant proteins we have shown that CL3 – the cytosolic loop between TM5 and TM6 – plays a key role in formation of the N‐terminal component of the glibenclamide binding site. Analysis of deletion variants of the C‐terminal part of SUR1 showed that CL8 – the cytosolic loop between TM15 and TM16 – is the only determinant for the C‐terminal component of the glibenclamide binding site. We suggest that in SUR1 in the native KATP channel close proximity of CL3 and CL8 leads to formation of the glibenclamide binding site.

Studies of the ATPase activity of the ABC protein SUR1

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.

Expression of functionally active ATP-sensitive K-channels in insect cells using baculovirus

We have expressed active ATP‐sensitive K‐channels (KATP charmels) in Spodoptera frugiperda (Sf9) cells using a baculovirus vector. A high yield of active channels was obtained on co‐infection with SUR1 and Kir6.2 engineered to contain N‐ and/or C‐terminal tags to permit detection by Western blotting. Channel activity was sensitive to ATP, glibenclamide and diazoxide. Channel activity was also obtained on expression of a C‐terminally truncated Kir6.2 (Kir6.2ΔC26): these channels were blocked by ATP but were insensitive to sulphonylureas. In contrast to Xenopus oocytes and mammalian cells the full length Kir6.2 also gave rise to active channels in Sf9 cells when expressed alone. The highest yield of active KATP channels was obtained on infection with a fusion protein containing SUR1 linked to Kir6.2ΔC26 via a 6‐amino acid linker.

BACULOVIRUS MULTIGENE EXPRESSION VECTORS AND THEIR USE FOR UNDERSTANDING THE ASSEMBLY PROCESS OF ARCHITECTURALLY COMPLEX VIRUS PARTICLES

The baculovirus expression vector is a eukaryotic DNA viral vector for the cloning and expression of foreign genes in cultured lepidopteran insect cells and insects. It has become an important tool for the large-scale production of recombinant proteins for a variety of applications including the structure-function analysis of genes and their gene products. We have developed a number of baculovirus multigene expression vectors and utilized these to understand the assembly process of multicomponent capsid structures of large viruses such as bluetongue virus (BTV), a member of the Orbivirus genus within the family Reoviridae. BTV is some 810 A in diameter and comprised of two protein shells containing four major proteins, VP2, VP5, VP7 and VP3, surrounding a genome of ten double-stranded RNA segments and three minor proteins (VP2, VP4 and VP6). BTV is the etiological agent of a sheep disease that is sometimes fatal in certain parts of the world (e.g., Africa, Asia, and the Americas). Using baculovirus multigene vectors, we have co-expressed various combinations of BTV genes in insect cells and produced structures that mimic the various stages of BTV assembly. For example, co-expressed VP3 and VP7 form BTV core-like particles, while co-expressed VP2, VP5, VP7 and VP3 form BTV virus-like particles. Using deletion, point and domain switching analyses of each protein, we have been able to identify certain sequences in the VP7 and VP3 proteins that are essential for the assembly of core-like particles. These expression and biochemical studies have been complemented by collaboration studies using cryo-electron microscopy and image processing analyses to provide the three-dimensional structure of the expressed particles. In addition and with other associates, we have used X-ray crystallography of VP7 to deduce its atomic structure. Extensive studies on the immune responses elicited by these self-assembled particles, and chimeric derivatives involving various foreign antigens, have been carried out. Finally, using as little as 10 microg of the self-assembled virus-like particles, we have shown that they can confer long-lasting protection in sheep against BTV.

The solution structure of the bovine leukaemia virus matrix protein and similarity with lentiviral matrix proteins

In the mature virion, retroviral matrix proteins are found in association with the inner face of the viral membrane. They play a critical role in determining the morphogenesis of virus assembly. We have determined the three‐dimensional solution structure of the bovine leukaemia virus (BLV) matrix protein by heteronuclear nuclear magnetic resonance. The protein contains four principal helices that are joined by short, partially structured loops. Despite no sequence similarity with the lentiviruses, the structure shows an intriguing homology with the equivalent protein from the human and simian immunodeficiency viruses. A root‐mean‐square deviation of 3.78 angstrom is observed over the backbone atoms of 36 equivalent helical positions. The similarity implies a possible common assembly unit for the matrix proteins of type C retroviruses.

Investigation of the molecular assembly of β‐cell KATP channels

We have investigated the protein interactions involved in the assembly of pancreatic β‐cell ATP‐sensitive potassium channels. The channels are a heterooligomeric complex of pore‐forming Kir6.2 subunits and sulfonylurea receptor (SUR1) subunits. SUR1 belongs to the ATP binding cassette (ABC) family of proteins and has two nucleotide binding domains (NBD1 and NBD2) and 17 putative transmembrane (TM) sequences. Previously we showed that co‐expression in a baculovirus expression system of two parts of SUR1 divided at Pro1042 between TM12 and 13 leads to restoration of glibenclamide binding activity, whereas expression of either individual N‐ or C‐terminal domain alone gave no glibenclamide binding activity [M.V. Mikhailov and S.J.H. Ashcroft (2000) J. Biol. Chem. 275, 3360–3364]. Here we show that the two half‐molecules formed by division of SUR1 between NBD1 and TM12 or between TM13 and 14 also self‐assemble to give glibenclamide binding activity. However, deletion of NBD1 from the N‐part of SUR1 abolished SUR1 assembly, indicating a critical role for NBD1 in SUR1 assembly. We found that differences in glibenclamide binding activity obtained after co‐expression of different half‐molecules are attributable to different amounts of binding sites, but the binding affinities remained nearly the same. Simultaneous expression of Kir6.2 resulted in enhanced glibenclamide binding activity only when the N‐half of SUR1 included TM12. We conclude that TM12 and 13 are not essential for SUR1 assembly whereas TM12 takes part in SUR1 Kir6.2 interaction. This interaction is specific for Kir 6.2 because no enhancement of glibenclamide binding was observed when half‐molecules were expressed together with Kir4.1. We propose a model of KATP channel organisation based on these data.

Morphological localisation of sulfonylurea receptor 1 in endocrine cells of human, mouse and rat pancreas

Aims/hypothesisSulfonylurea receptor 1 (SUR1) is the regulatory subunit of ATP-sensitive K channels in beta cells. Morphological methods (immunohistochemistry and sulfonylurea binding) were used to establish the cellular and subcellular location of SUR1 in human and rodent islets.ResultsIn the human, mouse and rat pancreas, all endocrine cells of the islets were immunolabelled with an anti-SUR1 antibody, whereas tissues containing SUR2 were consistently negative, as were those from Sur1 (also known as Abcc8)−/− mice. In beta cells of the three species, the plasma membrane was distinctly stained, but SUR1 was mainly present over the cytoplasm, with an intensity that varied between cells. Electron microscopy showed that SUR1 was immunolocalised in insulin, glucagon and somatostatin granules. In rat beta cells degranulated by in vivo treatment with glibenclamide (known as glyburide in the USA and Canada), the insulin and SUR1 staining intensity was similarly decreased by ∼45%, whereas SUR1 staining was not changed in non-beta cells. In all islet cells, binding of glibenclamide labelled with fluorescent dipyrromethane boron difluoride (BODIPY-FL) was punctate over the cytoplasm, compatible with the labelling of endocrine granules. A faint labelling persisted in Sur1−/− mice, but it was not different from that obtained with BODIPY-FL alone used as negative control.Conclusions/interpretationOur study immunolocalised SUR1 in alpha, beta and delta cells of human, mouse and rat islets, and for the first time visualised it in the plasma membrane. We also show that SUR1 is abundant in endocrine granules, where its function remains to be established. No specific sulfonylurea-binding sites other than SUR1 are identified in islet cells by the glibenclamide–BODIPY-FL technique.

A novel simple method for the investigation of drug binding to the KATP channel sulfonylurea receptor

H-labeledglibenclamide was bound to the column andeluted stepwise with increasing concentration ofunlabeled drug. The concentration of the drugcorresponding to the maximum of the elutioncurve was found to be characteristic for the drugand the proposed method can be used forscreening and rapid characterisation of new drugsthat have the same target as glibenclamide.