西亚试剂：：The Mitochondrial ROMK Channel is a Molecular

D. Brian Foster1, Alice S. Ho1, Jasma Rucker1, Anders O. Garlid2, Ling Chen1, Agnieszka Sidor1, Keith D. Garlid2 and Brian O'Rourke

Rationale: Activation of the mitochondrial ATP-sensitive potassium channel (mitoKATP) has been implicated in the mechanism of cardiac ischemic preconditioning, yet its molecular composition is unknown. Objective: To use an unbiased proteomic analysis of the mitochondrial inner membrane to identify the mitochondrial K+ channel underlying mitoKATP. Methods and Results: Mass spectrometric analysis was used to identify KCNJ1(ROMK) in purified bovine heart mitochondrial inner membrane and confirmed that ROMK mRNA is present in neonatal rat ventricular myocytes and adult hearts. ROMK2, a short form of the channel, is shown to contain an N-terminal mitochondrial targeting signal and a full length epitope-tagged ROMK2 colocalizes with mitochondrial ATP synthase β. The high-affinity ROMK toxin, tertiapin Q, inhibits mitoKATP activity in isolated mitochondria and in digitonin-permeabilized cells. Moreover, shRNA-mediated knockdown of ROMK inhibits the ATP-sensitive, diazoxide activated, component of mitochondrial thallium uptake. Finally, the heart-derived cell line, H9C2, is protected from cell death stimuli by stable ROMK2 overexpression, while knockdown of the native ROMK exacerbates cell death. Conclusions: The findings support ROMK as the pore-forming subunit of the cytoprotective mitoKATP channel.