Abstract 1289: FRET Analysis of ATP-Sensitive K+ Channels in Mitochondrial Inner Membrane
The ATP-sensitive K+ channels (KATP) in both sarcolemmal (sarcKATP) and mitochondrial inner membrane (mitoKATP) are the critical mediators in cellular protection of ischemic preconditioning (IPC). Whereas cardiac sarcKATP contains Kir6.2 and SUR2A, the molecular identity of mitoKATP remains inconclusive. Giving that most of mitochondrial proteins are imported into mitochondria from the cytosol, we tested the hypothesis that protein kinase C (PKC), a central mediator in IPC, may stimulate the insertion of KATP channels into mitochondrial inner membrane. The mitochondrial localization of Kir6.2-containing KATP channels was assessed by microscopic analysis of fluorescence resonance energy transfer (FRET). The KATP-deficient COS-7 cells were used to determine whether CFP fused to the C-terminus of Kir6.2 is sufficiently close to Mito-YFP, which is targeted to mitochondrial matrix, to yield FRET. Whereas no significant FRET signals were detected in cells without PMA treatment, cells exposed to PMA for 30 – 60 min exhibited FRET signals due to energy transfer from CFP at the C-terminus of Kir6.2 to YFP in the mitochondrial matrix. When normalized against Mito-YFP intensity, these FRET signals were smaller than the similarly normalized control FRET signal between Mito-CFP and Mito-YFP, likely a reflection of the lower amount of Kir6.2-CFP as compared to Mito-CFP in the control experiment. When normalized against the CFP intensity, the FRET signal between Mito-CFP and Mito-YFP was comparable to that between Kir6.2-CFP and Mito-YFP after PMA treatment for 60 min, indicating that by then the KATP channels targeted to the mitochondria had fully exposed the CFP at the C-terminus of Kir6.2 to the matrix. Similar FRET signals were observed in cells expressing Mito-CFP, SUR2A/Kir6.2-YFP. These observations were further supported by the microscopic and functional studies of KATP channels in mitochondria. For dipole-dipole coupling to induce energy transfer from donor to acceptor, the fluorophore at the N- or C-terminus of Kir6.2 has to be within 6–10 nm of the fluorophore within the mitochondrial matrix. These findings strongly suggest that PKC activation causes insertion of functional Kir6.2-containing KATP channels into the mitochondrial inner membrane.