FIGURE 4.
CaMKII effect in Kir6.2T224A and Kir6.2T180A mutants. A, in isolated recombinant cardiac KATP channels with WT or mutated Kir6.2, an in vitro phosphorylation assay was performed in the presence and absence of CaMKII. Kir6.2-HA subunits were mutated from threonine to alanine at Thr-224 or Thr-180 or both. Kir6.2-HA is identified as a double band at ∼37 kDa. The prominent signal near 50 kDA level represents autophosphorylated CaMKII. B, in WT and mutated peptide fragments of Kir6.2, in vitro phosphorylation assay was performed in the presence and absence of CaMKII. Fragments were mutated from threonine to alanine at Thr-224 or Thr-180. Differences in band migration are attributed to different sizes and charge of the fragments. The WT Thr-180 and mutant T180A fragments are identified at ∼3.3 kDa (best seen for Thr-180 + CaMKII and faintly for T180A + CaMKII), whereas the Thr-224 and T224A fragments are identified at ∼4.5 kDa (best seen for Thr-224 + CaMKII). C–H, representative immunofluorescence confocal imaging was performed in HEK cells following the same protocol as in Fig. 3. C and D, cells expressing CaMKII-GFP, Kir6.2-HA, and SUR2A are shown. E and F, cells expressing CaMKII-GFP, Kir6.2T180A-HA, and SUR2A are shown. G and H, cells expressing CaMKII-GFP, Kir6.2T224A-HA and SUR2A. I, summary data for immunofluorescence imaging is shown. For WT, n = 34 cells were from 2 transfections. For T224A, n = 4 transfections, 48 cells; *, p < 0.05 versus WT. For T180A, n = 4 transfections, 42 cells; *, p < 0.05 versus WT. J, representative whole cell KATP channel current was stimulated by 50 μmol/liter pinacidil (pin) and 50 μmol/liter DNP in a HEK cell expressing CaMKII-GFP, Kir6.2T224A, and SUR2A in response to A23187 5 μmol/liter. pF, picofarads. K, summary data of pinacidil- and DNP-stimulated whole cell KATP channel current in response to A23187 in HEK cells expressing CaMKII-GFP, SUR2A, and WT Kir6.2 versus Kir6.2T224A (*, p < 0.05).