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Open AccessArticle

Single-Channel Properties of the ROMK-Pore-Forming Subunit of the Mitochondrial ATP-Sensitive Potassium Channel

1
Laboratory of Intracellular Ion Channels, Nencki Institute of Experimental Biology, Polish Academy of Sciences, 02-093 Warsaw, Poland
2
Department of Physics and Biophysics, Institute of Biology, Warsaw University of Life Sciences-SGGW, 02-787 Warsaw, Poland
3
Division of Cardiology, The Johns Hopkins University School of Medicine, 720 Rutland Avenue, Baltimore, MD, USA
*
Author to whom correspondence should be addressed.
Present address: Department of Biosciences, University of Milan, 20133 Milan, Italy.
Present address: Department of BioMedical Research, University of Bern/Department of Nephrology and Hypertension, Inselspital Bern, Bühlstrasse 28, CH-3010 Bern, Switzerland.
Int. J. Mol. Sci. 2019, 20(21), 5323; https://doi.org/10.3390/ijms20215323
Received: 28 September 2019 / Revised: 21 October 2019 / Accepted: 23 October 2019 / Published: 25 October 2019
(This article belongs to the Special Issue Ion Channel and Ion-Related Signaling 2019)
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. View Full-Text
Keywords: mitochondria; patch-clamp; cardiac muscle; mitochondrial ATP-sensitive potassium channel; renal outer medullary potassium channel; mitochondrial large conductance calcium regulated potassium channel mitochondria; patch-clamp; cardiac muscle; mitochondrial ATP-sensitive potassium channel; renal outer medullary potassium channel; mitochondrial large conductance calcium regulated potassium channel
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Laskowski, M.; Augustynek, B.; Bednarczyk, P.; Żochowska, M.; Kalisz, J.; O’Rourke, B.; Szewczyk, A.; Kulawiak, B. Single-Channel Properties of the ROMK-Pore-Forming Subunit of the Mitochondrial ATP-Sensitive Potassium Channel. Int. J. Mol. Sci. 2019, 20, 5323.

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