Mitochondrial ROMK Channel Is a Molecular Component of MitoK _ATP

Abstract

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.

Early opening of sarcolemmal ATP-sensitive potassium channels is not a key step in PKC-mediated cardioprotection

“…Pharmacological evidence, using the classical mitoK ATP channel agents diazoxide and 5-HD, would suggest that mitoK ATP channels do indeed play a role; however the specificity of these agents has been called into question [23]. The recent identification of ROMK as a putative mitoK ATP channel subunit [24] provides a potential target for further investigation. Additionally, the role of sarcoK ATP channels in cardioprotection may be as part of a larger signalasome-type complex [40].…”

ATP synthase K+- and H+-flux drive ATP synthesis and enable mitochondrial K+-uniporter function

“…Using an electrophysiological approach, we ruled out the presence of any other cation-selective channel activity in our F1Fo preparations (Figure 1 -figure supplement 2A,B). It has been suggested that a mitochondrial ROMK potassium channel might act as the pore-forming subunit of a cytoprotective mKATP channel (Foster et al, 2012). Our immunoblotting with anti-ROMK antibody ruled out ROMK channel contamination of the isolated F1Fo (Figure 1 -figure supplement 2C).…”

“…Nature usually operates important pathways with built-in redundancy so that other mitochondrial K + channels may contribute to these mechanisms, including the Ca 2+ -activated K + channel, BKCa (Xu et al, 2002), and a ROMK channel which may also mimic mKATP channel function (Foster et al, 2012), but these pathways are likely fine-tuning mechanisms.…”

Diazoxide Cardioprotection Is Independent of Adenosine Triphosphate-Sensitive Potassium Channel Kir 6.1 Subunit in Response to Stress

“…Diazoxide does not elicit sK ATP channel current in ventricular myocytes, and its protective benefits require the presence of the K ATP channel subunit SUR1 and may involve inhibition of succinate dehydrogenase (15,18,19). Activity at a proposed mitochondrial K ATP channel is supported only by indirect methods and no mitochondrial K ATP channel has been identified at the molecular level or cloned (23,24). Similarly, efforts to localize known K ATP subunits in mitochondria have been inconsistent and controversial (25,26).…”