Regulation of the Activity of Mitochondrial Large-Conductance Calcium-Activated Potassium Channel Through a New External Heme-Binding Site

Abstract

Mitochondrial large-conductance calcium-activated potassium (mitoBKCa) channel located in the inner mitochondrial membrane is formed by the DEC splice variant of the KCNMA1 gene. The activity of mitoBKCa is regulated by many modulators, including activators such as calcium ions and inhibitors such as heme and its oxidized form hemin. Heme/hemin binds to the heme-binding motif (–CXXCH–) located between two RCK (regulating conductance of K+) domains present in the mitochondrial matrix (internal side of the channel). Till now, there was no evidence about the existence of another heme-binding site in the mitoBKCa. However, using patch-clamp on mitoplast isolated from astrocytoma U-87MG cells and HEK293 cell line stably expressing DEC isoform of BKCa, we accidentally found that external heme also inhibits mitoBKCa suggesting the presence of yet another unknown heme-binding site. To further confirm the initial observation we used two approaches. First, we carried out patch-clamp recordings on the inner mitochondrial membrane in the outside-out configuration, and second, the pipette perfusion system was used in the inside-out configuration of the membrane patches. Both approaches allow for the application of channel modulators to the intermembrane-space (external) side of the mitoBKCa. We confirmed that hemin applied to the intermembrane space side of the channel inhibits the activity of mitoBKCa. We investigated the amino acid composition of different isoforms of BKCa and identified cysteines and histidine located at the external side of the channel, which could constitute a novel heme-binding site. We obtained HEK293 cell lines stably expressing DEC mutants of the above-mentioned cysteines and histidine. Patch-clamp experiments will be carried out to find out whether introduced mutations affect channel inhibition by external heme. This work was supported by the Polish National Science Center, grant no. 2018/31/N/NZ1/00928.