Mitochondrial ROMK channel is a molecular component of mitoKATP

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 ROMK mRNA was confirmed to be present in neonatal rat ventricular myo _cytes 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, short hairpin RNA-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, whereas knockdown of the native ROMK exacerbates cell death. CONCLUSIONS:: The findings support ROMK as the pore-forming subunit of the _cytoprotective mitoKATP channel.