Investigation of the molecular assembly of β‐cell KATP channels

FEBS Letters

Mikhailov, Michael V¹  Mikhailova, Ellina A¹  Ashcroft, Stephen J.H¹ 

[1] Nuffield Department of Clinical Laboratory Sciences, John Radcliffe Hospital, Headington, Oxford OX3 9DU, UK

关键词: Potassium channel;    Sulfonylurea receptor;    Baculovirus;    ABC protein;   

DOI  :  10.1016/S0014-5793(00)02035-4

学科分类：生物化学/生物物理

来源: John Wiley & Sons Ltd.

【 摘 要 】

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 K_ATP channel organisation based on these data.

【 授权许可】

Unknown