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Characterization of interaction sites between Kir6.0 and SUR subunits of ATP-sensitive potassium (Katp) channels

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posted on 2014-12-15, 10:34 authored by Mohammed Aljohi
This study investigated cytoplasmic inter-subunit interactions between the Kir6.2 and SUR2A subunits of the cardiac ATP-sensitive potassium channel. The channels are a heterooligomeric complex of pore-forming Kir6.2 subunits and sulphonylurea receptor (SUR2A) subunits. Interactions between the cytoplasmic loops, the nucleotide binding domains (NBF1 and NBF2) of SUR2A and the full length of Kir6.2 were determined. In co-immunoprecipitation experiments, fragments from the C-terminal of SUR2A containing residues 1294-1358 tagged with Maltose-binding protein (MBP) showed binding with the full length Kir6.2 subunit, while residues between 1358-1545 did not. This indicated involvement of a 65 amino acid domain in the proximal C-terminal of SUR2A in forming a direct interaction with Kir6.2. When HEK 293 cells stably expressing Kir6.2/SUR2A channels were transiently transfected with SUR2A fragments containing residues 1294-1359, KATP current was decreased. This current reduction was due to a decreased number of channel subunits in the cell membrane; this was demonstrated by using immunocytochemistry, which showed that anti-K ATP channel subunit-associated fluorescence was lower in the cell membrane and increased in the intracellular compartment in the presence of the binding region.;Use of SUR2A/MRP1 chimaeras of the putative binding domain showed that the last eleven amino acids of the binding region were important for binding activity but that they do not contain all the elements necessary for binding. Co-immunoprecipitation and assays of disruption of functional channels with the binding domain chimaeras suggested an important role for the residues between 1318 and 1337 in the Kir6.2 binding motif within the SUR2A C-terminal domain.

History

Date of award

2005-01-01

Author affiliation

Cardiovascular Sciences

Awarding institution

University of Leicester

Qualification level

  • Doctoral

Qualification name

  • PhD

Language

en

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