Biomedical subjects

E A Mikhailova

Publications and source records attributed to E A Mikhailova.

3 recordsLinked to original sources

PubMed2002

Structure-function relationships in the beta-cell K(ATP) channel.

The ATP-sensitive potassium (K(ATP)) channel plays a key role in controlling beta-cell membrane potential and insulin secretion. The channels are composed of two subunits, Kir6.2, which forms the channel pore, and SUR1, which contains binding sites for nucleotides and sulphonylureas and acts as a channel regulator. Our current studies are aimed at delineating the molecular interactions involved in assembly and ligand binding by K(ATP) channel proteins. We have employed a complementation approach in which SUR1 half-molecules are co-expressed in insect cells using a baculovirus system. Together with data from truncated SUR1 molecules and a fusion protein in which SUR1 is linked to Kir6.2, we have interpreted our findings in terms of a model for the structure of the K(ATP) channel. The main features of the model are: (i) the C-terminal end of SUR1 is close to the N-terminus of Kir6.2; (ii) the two nucleotide binding domains (NBDs) of SUR1--NBD1 and NBD2--are in proximity; (iii) transmembrane helix 12 of SUR1 is orientated in such a way that it can make contact with Kir6.2; (iv) formation of the glibenclamide binding site requires that the two cytosolic loops (CLs) CL3 and CL8 are located close to each other; (v) there are homomeric interactions between the NBD1 domains of neighbouring subunits. We suggest that binding of glibenclamide leads to conformational changes in CL3 and CL8 leading to rearrangement of transmembrane helices. These effects are transmitted to Kir6.2 to result in channel closure.

ATP-Binding Cassette Transporters↗

PubMed2001

Molecular structure of the glibenclamide binding site of the beta-cell K(ATP) channel.

We have investigated the structure of the glibenclamide binding site of pancreatic beta-cell ATP-sensitive potassium (K(ATP)) channels. K(ATP) 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 K(ATP) channel close proximity of CL3 and CL8 leads to formation of the glibenclamide binding site.

ATP-Binding Cassette Transporters↗

PubMed2000

Investigation of the molecular assembly of beta-cell K(ATP) channels.

We have investigated the protein interactions involved in the assembly of pancreatic beta-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 K(ATP) channel organisation based on these data.

ATP-Binding Cassette Transporters↗