A Cytoplasmic Inter-Subunit Salt Bridge, Kir6.1R347/SUR2AE1318, Contributes to Allosteric Information Transmission in Kir6.14/SUR2A4 Channel Complexes

KATP channels are hetero-octamers composed of pore forming Kir6 (Kir6.1 or Kir6.2) and a regulatory sulphonylurea receptor (SUR1, SUR2A or SUR2B) subunits. Previous studies have revealed a salt bridge between Kir6.2K338 and SUR2AE1318 that is involved in inter-subunit transmission of allosteric information (Rubaiy et al, Biophysical J. 2011: 100; P432a). The aim of this study was to assess whether Kir6.1R347, which corresponds to Kir6.2K338, plays a similar role in Kir6.1-containing channels.Whole-cell patch clamp recording was employed to assess channel sensitivity to pinacidil and glibenclamide following mutation of single residues or reinstatement of the proposed salt bridge by paired charge reversals in full length Kir6.1/SUR2A channel subunits after heterologous expression in HEK-293 cells. A single point mutation, Kir6.1R347E expressed with wild type SUR2A increased significantly (p < 0.006) the sensitivity to activation by pinacidil of Kir6.1R347E/SUR2AWT channels (EC50 = 0.71 ± 1.21 µM) versus wild type Kir6.1/SUR2A channels (EC50 = 43.90 ± 1.28 µM). Reinstatement of the cytoplasmic electrostatic interaction in the Kir6.1R347E/SUR2AE1318R subunit combination reversed the sensitivity to pinacidil to near wild type (EC50 = 23.5 ± 1.3 µM, p < 0.028). Furthermore, glibenclamide sensitivity was reduced significantly in the Kir6.1R374E/SUR2AWT channel (IC50 = 241 ± 1.09 nM, p < 0.015) and restored in Kir6.1R347E/SUR2AE1318R (IC50 = 13.75 ± 1.11 nM, p < 0.080) versus wild type Kir6.1/SUR2A channel (IC50 = 6.14 ± 1.13 nM). These data indicate that, like Kir6.2K338, Kir6.1R347 makes a crucial contribution to allosteric information transmission from SUR2A to the channel pore through inter-subunit salt bridge formation with SUR2A1318.