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 (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.

Diazoxide affects mitochondrial bioenergetics by the opening of mKATP channel on submicromolar scale

“…As it was reported in earlier studies, in the heart, Kir6.2 was dispensable for the K + conductance stimulated by diazoxide [31]. Also, none of ROMK isoforms (Kir1.1, Kir3.1, and Kir3.4) proposed as K + conductant subunit of mK ATP -channel [33,34] was responsible for diazoxide-induced swelling of cardiac mitochondria [32]. These studies indicated that K + conductant subunit of mK ATP -channel didn't belong to either Kir6.x, or Kir1.x channels, but molecular nature of the K + pore of mK ATP -channel remained elusive.…”

Mitochondrial Potassium Channels as Druggable Targets

“…ROMK proteins are encoded by distinct splice variants of the Kcnj1 gene. The expression of ROMK in mitochondria and patch-clamp measurements confirmed its functional properties as a mitoK ATP channel [27]. Recently, it was proposed that the CCDC61 and ABCB8 proteins constitute a mitoK ATP channel composed of pore-forming and ATP-binding subunits (mitochondrial sulfonylurea receptor) [29].…”

“…The current (June 2020) list of identified mitochondrial potassium channels is as follows: mitoK ATP channel: mitochondrial ATP-regulated potassium channel, the first potassium channel described in the inner mitochondrial membrane [ 1 ]. Most likely, these channels might be formed by two proteins: ROMK-type channels (encoded by KCNJ1 gene) [ 25 , 26 , 27 , 28 ] or the recently described CCDC51 protein (encoded by CCDC51 gene) [ 29 ]. Additionally, ABCB8 protein (mitochondrial sulfonylurea receptor) is part of the mitoK ATP channel [ 29 ].…”

Experimental assessment of carbacetam effect on the cerebral mitochondria in rats with scopolamine-induced Alzheimer’s disease

“…Moreover, carbacetam is not excluded to modulate Ca 2+ and K + currents. Activation of mitochondrial ATP-dependent potassium channels is known to result in the decrease of Ca 2+ load and inhibition of an excessive opening of the mitochondrial pore 16,17 .…”