Functional ATP-sensitive potassium (KATP) channels are a hetero-octomer of four inward rectifier Kir6.0 and four sulphonylurea receptor (SUR) subunits. Replacement of the transmembrane domains TMD0, TMD1 or TMD2 in SUR1 with those from multidrug resistance-associated protein 1 impairs assembly and surface expression of KATP channels (Schwappach, 2000). In this study, possible interactions between the carboxy-terminal domain of SUR2A and Kir6.2 were investigated by co-immunoprecipitation of maltose binding protein (MBP)-tagged SUR2A fragments with Kir6.2 and by analysis of cloned KATP channel function and distribution in HEK293 cells.
KATP channel polypeptides were prepared by in vitro transcription/translation of cloned cDNA. Two MBP-tagged SUR2A fragments A (amino acids 1254-1545) and B (amino acids 1294-1403) were co-immunoprecipitated with full length Kir6.2 using a polyclonal anti-Kir6.2 antiserum. A third C-terminal domain fragment, C (amino acids 1358-1545) did not co-immunoprecipitate with Kir6.2 under the same conditions, thus indicating a direct interaction between Kir6.2 and a cytoplasmic carboxy-terminal region of SUR2A between residues 1294 and 1358.
HEK293 cells stably expressing full length Kir6.2 and SUR2A were transiently transfected with SUR2A fragments cloned into pIRES2-EGFP. Fractional ATP and glibenclamide-sensitive currents were reduced in inside-out patches from cells transfected with fragments A (0.050 ± 0.004, mean ± S.E.M., P < 0.001) and B (0.13 ± 0.02, P < 0.001) compared with mock transfected and fragment C (0.93 ± 0.10, P > 0.05) transfected cells (control current = 1283 ± 182 pA (S.E.M.), n = 6, unpaired t test with Bonferroni’s multiple comparison test), suggesting a reduced number of functional channels at the cell surface. In mock transfected cells, immunocytochemistry with isoform-specific anti-Kir6.2 and anti-SUR2A antisera revealed a predominant plasma membrane localisation of both subunits. Cell membrane-associated fluorescence was substantially lower and intracellular fluorescence was raised in cells expressing fragments A and B compared with untreated cells or cells expressing fragment C. Thus SUR2A fragments containing residues 1294-1358 reduce current by decreasing channel subunits in the cell membrane.
We have identified a site in the carboxy-terminal domain of SUR2A, between residues 1294 and 1358, whose direct interaction with full length Kir6.2 is crucial for assembly of functional KATP channels.
This work was funded by the BHF and MRC. We thank A. Tinker for Kir6.2/SUR2A cell lines.