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

ABSTRACT: An increased flux of potassium ions into the mitochondrial matrix through the ATP-sensitive potassium channel (mitoK_ATP) 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 mitoK_ATP in heart mitochondria. Our research focuses on the properties of mitoK_ATP from heart-derived H9c2 cells. For the first time, we detected single-channel activity and describe the pharmacology of mitoK_ATP 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 mitoK_ATP. ROMK2 overexpression resulted in a significant increase of mitoK_ATP 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 mitoK_ATP inhibitor) and by Tertiapin Q (an inhibitor of both the ROMK-type channels and mitoK_ATP). Additionally, mitoK_ATP 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 mitoK_ATP.

SUBMITTER: Laskowski M 

PROVIDER: S-EPMC6862428 | biostudies-literature | 2019 Oct

REPOSITORIES: biostudies-literature