Figure 8.

Increased ATP sensitivity in channels formed by E203K-SUR1 and Q52E-Kir6.2 compromise their ability to open in response to metabolic inhibition. (A and B) Processing and surface expression of K_ATP channels were assessed in COSm6 cells transfected with WT or mutant channels. (A) Western blot of SUR1 using anti-SUR1 antibody as described in Materials and methods. The complex-glycosylated, mature form of SUR1 (a proxy for surface expression) is indicated by the closed arrowhead, and the core-glycosylated, immature form is indicated by the open arrowhead. Mutations at Q52E-Kir6.2 and/or E203K-SUR1 do not disrupt protein trafficking. (B) K_ATP surface expression was quantified using chemiluminescence assays. Mutations at Q52E-Kir6.2 or E203K-SUR1 or both do not affect the number of channels at the cell surface. Control F55L-Kir6.2 channels also have WT-like surface expression (Lin et al., 2006b). Error bars represent SEM; n = 3. (C) Mean ⁸⁶Rb⁺ efflux profile from COSm6 cells transiently expressing WT or mutant channel subunits. Efflux was measured in Ringer’s solution after metabolic inhibition (i.e., decreased intracellular ATP production). Channels composed of Q52E-Kir6.2 with and without E203K-SUR1 show significantly diminished efflux compared with controls, WT and F55L-Kir6.2; the latter control was included because it has decreased intrinsic P_o (∼0.11) but normal ATP sensitivity and surface expression. Error bars represent SEM for n = 3 experiments, except F55L-Kir6.2, which represents the difference between n = 2 experiments. Some error bars are smaller than the symbols.