Mitochondrial ROMK channel is a molecular component of mitoKATP

Darren Brian Foster, Alice S. Ho, Jasma Rucker, Anders O. Garlid, Ling Chen, Agnieszka Sidor, Keith D. Garlid, Brian O'Rourke

Research output: Contribution to journalArticle

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 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.

Original languageEnglish (US)
Pages (from-to)446-454
Number of pages9
JournalCirculation Research
Volume111
Issue number4
DOIs
StatePublished - Aug 3 2012

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Mitochondrial Membranes
Cell Death
Mitochondrial Proton-Translocating ATPases
Diazoxide
Digitonin
KATP Channels
Ischemic Preconditioning
Thallium
Proteomics
Small Interfering RNA
Epitopes
Mitochondria
Adenosine Triphosphate
Cell Line
Messenger RNA
mitochondrial K(ATP) channel
tertiapin

Keywords

  • oprotection
  • apoptosis
  • ATP-sensitive potassium channel
  • ischemia
  • mitochondria
  • reconditioning
  • renal outer medullary potassium channel

ASJC Scopus subject areas

  • Physiology
  • Cardiology and Cardiovascular Medicine

Cite this

Mitochondrial ROMK channel is a molecular component of mitoKATP. / Foster, Darren Brian; Ho, Alice S.; Rucker, Jasma; Garlid, Anders O.; Chen, Ling; Sidor, Agnieszka; Garlid, Keith D.; O'Rourke, Brian.

In: Circulation Research, Vol. 111, No. 4, 03.08.2012, p. 446-454.

Research output: Contribution to journalArticle

Foster, DB, Ho, AS, Rucker, J, Garlid, AO, Chen, L, Sidor, A, Garlid, KD & O'Rourke, B 2012, 'Mitochondrial ROMK channel is a molecular component of mitoKATP', Circulation Research, vol. 111, no. 4, pp. 446-454. https://doi.org/10.1161/CIRCRESAHA.112.266445
Foster, Darren Brian ; Ho, Alice S. ; Rucker, Jasma ; Garlid, Anders O. ; Chen, Ling ; Sidor, Agnieszka ; Garlid, Keith D. ; O'Rourke, Brian. / Mitochondrial ROMK channel is a molecular component of mitoKATP. In: Circulation Research. 2012 ; Vol. 111, No. 4. pp. 446-454.
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AU - Sidor, Agnieszka

AU - Garlid, Keith D.

AU - O'Rourke, Brian

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AB - 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.

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