Abstract 17423: The Cardioprotective MitoK&rtf-inf-start;ATP&rtf-inf-end; Channel - Unmasked
Rationale: Activation of the mitochondrial ATP-sensitive potassium channel (MitoKATP) plays a critical role in cardiac ischemic preconditioning, yet the molecular identity of the channel subunits remains unknown. Objective: To use proteomic analysis of the mitochondrial inner membrane to identify the mitochondrial K+ channel underlying MitoKATP activity.
Results: A comprehensive proteomic approach led to the identification of KCNJ1(ROMK) in the inner membrane membrane of the bovine heart by mass spectrometry. Expression of ROMK in tissues that harbor MitoKATP activity, including the heart, liver and brain, was confirmed by PCR. Bioinformatic analysis suggested that one isoform, ROMK2, was the most likely to be targeted to mitochondria, and that the first 24 amino acids might constitute a bona fide mitochondrial targeting sequence. These predictions were confirmed when fusion of the N-terminus of ROMK2 to GFP conferred co-localization with mitochondrially- targeted fluorescent probe, TMRM in neonatal rat ventricular myocytes (NRVMs). Moreover, full length epitope-tagged ROMK2 also colocalized with the mitochondrial marker, ATP synthase beta. MitoKATP activity in isolated mitochondria and in digitonin-permeabilized cells was inhibited by the high-affinity ROMK toxin, Tertiapin-Q, and knockdown of endogenous ROMK expression by shRNA inhibited the ATP-sensitive, diazoxide-activated, component of mitochondrial thallium uptake. Importantly, the heart-derived H9C2 cell line is protected from oxidative stress-induced cell death by stable ROMK2 overexpression. Conversely, ablation of ROMK exacerbates cell death. This study extends the initial work by identifying ROMK-binding partners, to define, in detail, the structure of the channel complex and potential regulatory proteins. Combining immunopurification of ROMK from HEK293 cells with quantitative mass spectrometry, has yielded an initial round of targets that are currently being rigorously validated. Finally, identification of more ROMK peptides will facilitate protein quantification in a variety of tissues using multiple reaction monitoring.
Conclusions: The data suggest mitochondrial ROMK underlies the activity of the long-sought cytoprotective MitoKATP channel.
- © 2012 by American Heart Association, Inc.