Single-channel properties of the ROMK-pore-forming subunit of the mitochondrial ATP-sensitive potassium channel

Michał Laskowski, Bartłomiej Augustynek, Piotr Bednarczyk, Monika Żochowska, Justyna Kalisz, Brian O’rourke, Adam Szewczyk, Bogusz Kulawiak

Research output: Contribution to journalArticle

Abstract

An increased flux of potassium ions into the mitochondrial matrix through the ATP-sensitive potassium channel (mitoKATP) has been shown to provide protection against ischemia-reperfusion injury. Recently, it was proposed that the mitochondrial-targeted isoform of the renal outer medullary potassium channel (ROMK) protein creates a pore-forming subunit of mitoKATP in heart mitochondria. Our research focuses on the properties of mitoKATP from heart-derived H9c2 cells. For the first time, we detected single-channel activity and describe the pharmacology of mitoKATP in the H9c2 heart-derived cells. The patch-clamping of mitoplasts from wild type (WT) and cells overexpressing ROMK2 revealed the existence of a potassium channel that exhibits the same basic properties previously attributed to mitoKATP. ROMK2 overexpression resulted in a significant increase of mitoKATP activity. The conductance of both channels in symmetric 150/150 mM KCl was around 97 ± 2 pS in WT cells and 94 ± 3 pS in cells overexpressing ROMK2. The channels were inhibited by 5-hydroxydecanoic acid (a mitoKATP inhibitor) and by Tertiapin Q (an inhibitor of both the ROMK-type channels and mitoKATP). Additionally, mitoKATP from cells overexpressing ROMK2 were inhibited by ATP/Mg2+ and activated by diazoxide. We used an assay based on proteinase K to examine the topology of the channel in the inner mitochondrial membrane and found that both termini of the protein localized to the mitochondrial matrix. We conclude that the observed activity of the channel formed by the ROMK protein corresponds to the electrophysiological and pharmacological properties of mitoKATP.

Original languageEnglish (US)
Article number5323
JournalInternational journal of molecular sciences
Volume20
Issue number21
DOIs
StatePublished - Nov 1 2019

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KATP Channels
adenosine triphosphate
Adenosinetriphosphate
Potassium
porosity
cells
Proteins
proteins
inhibitors
Mitochondria
pharmacology
Potassium Channels
ischemia
Assays
mitochondria
Topology
matrices
Fluxes
Membranes
Acids

Keywords

  • Cardiac muscle
  • Mitochondria
  • Mitochondrial ATP-sensitive potassium channel
  • Mitochondrial large conductance calcium regulated potassium channel
  • Patch-clamp
  • Renal outer medullary potassium channel

ASJC Scopus subject areas

  • Catalysis
  • Molecular Biology
  • Spectroscopy
  • Computer Science Applications
  • Physical and Theoretical Chemistry
  • Organic Chemistry
  • Inorganic Chemistry

Cite this

Single-channel properties of the ROMK-pore-forming subunit of the mitochondrial ATP-sensitive potassium channel. / Laskowski, Michał; Augustynek, Bartłomiej; Bednarczyk, Piotr; Żochowska, Monika; Kalisz, Justyna; O’rourke, Brian; Szewczyk, Adam; Kulawiak, Bogusz.

In: International journal of molecular sciences, Vol. 20, No. 21, 5323, 01.11.2019.

Research output: Contribution to journalArticle

Laskowski, Michał ; Augustynek, Bartłomiej ; Bednarczyk, Piotr ; Żochowska, Monika ; Kalisz, Justyna ; O’rourke, Brian ; Szewczyk, Adam ; Kulawiak, Bogusz. / Single-channel properties of the ROMK-pore-forming subunit of the mitochondrial ATP-sensitive potassium channel. In: International journal of molecular sciences. 2019 ; Vol. 20, No. 21.
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abstract = "An increased flux of potassium ions into the mitochondrial matrix through the ATP-sensitive potassium channel (mitoKATP) has been shown to provide protection against ischemia-reperfusion injury. Recently, it was proposed that the mitochondrial-targeted isoform of the renal outer medullary potassium channel (ROMK) protein creates a pore-forming subunit of mitoKATP in heart mitochondria. Our research focuses on the properties of mitoKATP from heart-derived H9c2 cells. For the first time, we detected single-channel activity and describe the pharmacology of mitoKATP in the H9c2 heart-derived cells. The patch-clamping of mitoplasts from wild type (WT) and cells overexpressing ROMK2 revealed the existence of a potassium channel that exhibits the same basic properties previously attributed to mitoKATP. ROMK2 overexpression resulted in a significant increase of mitoKATP activity. The conductance of both channels in symmetric 150/150 mM KCl was around 97 ± 2 pS in WT cells and 94 ± 3 pS in cells overexpressing ROMK2. The channels were inhibited by 5-hydroxydecanoic acid (a mitoKATP inhibitor) and by Tertiapin Q (an inhibitor of both the ROMK-type channels and mitoKATP). Additionally, mitoKATP from cells overexpressing ROMK2 were inhibited by ATP/Mg2+ and activated by diazoxide. We used an assay based on proteinase K to examine the topology of the channel in the inner mitochondrial membrane and found that both termini of the protein localized to the mitochondrial matrix. We conclude that the observed activity of the channel formed by the ROMK protein corresponds to the electrophysiological and pharmacological properties of mitoKATP.",
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AU - Augustynek, Bartłomiej

AU - Bednarczyk, Piotr

AU - Żochowska, Monika

AU - Kalisz, Justyna

AU - O’rourke, Brian

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AB - An increased flux of potassium ions into the mitochondrial matrix through the ATP-sensitive potassium channel (mitoKATP) has been shown to provide protection against ischemia-reperfusion injury. Recently, it was proposed that the mitochondrial-targeted isoform of the renal outer medullary potassium channel (ROMK) protein creates a pore-forming subunit of mitoKATP in heart mitochondria. Our research focuses on the properties of mitoKATP from heart-derived H9c2 cells. For the first time, we detected single-channel activity and describe the pharmacology of mitoKATP in the H9c2 heart-derived cells. The patch-clamping of mitoplasts from wild type (WT) and cells overexpressing ROMK2 revealed the existence of a potassium channel that exhibits the same basic properties previously attributed to mitoKATP. ROMK2 overexpression resulted in a significant increase of mitoKATP activity. The conductance of both channels in symmetric 150/150 mM KCl was around 97 ± 2 pS in WT cells and 94 ± 3 pS in cells overexpressing ROMK2. The channels were inhibited by 5-hydroxydecanoic acid (a mitoKATP inhibitor) and by Tertiapin Q (an inhibitor of both the ROMK-type channels and mitoKATP). Additionally, mitoKATP from cells overexpressing ROMK2 were inhibited by ATP/Mg2+ and activated by diazoxide. We used an assay based on proteinase K to examine the topology of the channel in the inner mitochondrial membrane and found that both termini of the protein localized to the mitochondrial matrix. We conclude that the observed activity of the channel formed by the ROMK protein corresponds to the electrophysiological and pharmacological properties of mitoKATP.

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