Large-conductance Ca²⁺-activated potassium channel in mitochondria of endothelial EA.hy926 cells

Abstract

Bednarczyk P, Koziel A, Jarmuszkiewicz W, Szewczyk A. Large-conductance Ca 2ϩ -activated potassium channel in mitochondria of endothelial EA.hy926 cells. Am J Physiol Heart Circ Physiol 304: H1415-H1427, 2013. First published March 29, 2013 doi:10.1152/ajpheart.00976.2012In the present study, we describe the existence of a large-conductance Ca 2ϩ -activated potassium (BKCa) channel in the mitochondria of the human endothelial cell line EA.hy926. A single-channel current was recorded from endothelial mitoplasts (i.e., inner mitochondrial membrane) using the patch-clamp technique in the mitoplast-attached mode. A potassiumselective current was recorded with a mean conductance equal to 270 Ϯ 10 pS in a symmetrical 150/150 mM KCl isotonic solution. The channel activity, which was determined as the open probability, increased with the addition of calcium ions and the potassium channel opener NS1619. Conversely, the activity of the channel was irreversibly blocked by paxilline and iberiotoxin, BKCa channel inhibitors. The open-state probability was found to be voltage dependent. The substances known to modulate BKCa channel activity influenced the bioenergetics of mitochondria isolated from human endothelial EA.hy926 cells. In isolated mitochondria, 100 M Ca 2ϩ , 10 M NS1619, and 0.5 M NS11021 depolarized the mitochondrial membrane potential and stimulated nonphosphorylating respiration. These effects were blocked by iberiotoxin and paxilline in a potassiumdependent manner. Under phosphorylating conditions, NS1619-induced, iberiotoxin-sensitive uncoupling diverted energy from ATP synthesis during the phosphorylating respiration of the endothelial mitochondria. Immunological analysis with antibodies raised against proteins of the plasma membrane BKCa channel identified a poreforming ␣-subunit and an auxiliary ␤2-subunit of the channel in the endothelial mitochondrial inner membrane. In conclusion, we show for the first time that the inner mitochondrial membrane in human endothelial EA.hy926 cells contains a large-conductance calciumdependent potassium channel with properties similar to those of the surface membrane BKCa channel. mitochondria; endothelium; potassium channel; electrophysiology; bioenergetics BECAUSE ATP SUPPLIES in endothelial cells are relatively independent of mitochondrial oxidative pathways, studies on the bioenergetics of endothelial mitochondria have not been intensive to date. However, several recent observations suggest that endothelial mitochondria not only contribute to ATP generation but are also centrally involved in maintaining the fine regulatory balance among mitochondrial calcium concentrations, reactive oxygen species (ROS) production, and nitric oxide production (9, 10, 16). Endothelial mitochondria may also function as sensors of alternations in the local environment and contribute to the survival of endothelial cells under oxidative stress, and the mitochondrial ROS of endothelial cells are important signaling molecules (43).Endothelial cells are involved in many aspects of vascular ...

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“…The application of paxilline to the pipette control solution led to irreversible inhibition of the channel activity. Taken together, our data indicate that the observed single channel activity is similar to that of the mitoBK Ca channel previously reported in glioma, cardiac, skeletal muscle and endothelium mitochondria (Siemen et al 1999, Xu et al 2002, Bednarczyk et al 2013a). …”

“…The activity of the putative mitochondrial BK Ca channel (mitoBK Ca ), thought to be stimulated by Ca 2+ and blocked by charybdotoxin, was initially detected in mitoplasts from a glioma cell line (Siemen et al 1999). Later, similar single-channel recordings were performed for the inner membranes of heart, skeletal muscle, brain and endothelium mitochondria (Xu et al 2002, Skalska et al 2008, Douglas et al 2006, Fahnik-Babaei et al 2011, Bednarczyk et al 2013a). The BK Ca channel opener NS1619 was found to increase K + influx of intact heart mitochondria in a way that was sensitive to BK Ca channel blockers, charybdotoxin and Iberiotoxin (Xu et al 2002).…”

Ca²⁺-Activated K⁺ Channels as Therapeutic Targets for Myocardial and Vascular Protection

“…44 In addition, multiple groups have documented increased respiration following BKCa activation using NS1619 and NS11021. 41, 46 However, in endothelial cells, iberiotoxinsensitive NS1619-induced respiration was associated with increased non-phosphorylating respiration or proton leak. 46 In addition, deletion of the channel was shown to impair mitochondrial respiration and reduce respiratory control ratios in mitochondria isolated from BKCa KO mice.…”

“…41, 46 However, in endothelial cells, iberiotoxinsensitive NS1619-induced respiration was associated with increased non-phosphorylating respiration or proton leak. 46 In addition, deletion of the channel was shown to impair mitochondrial respiration and reduce respiratory control ratios in mitochondria isolated from BKCa KO mice. 8 Our group has recently demonstrated enhanced respiration by rottlerin following in vitro hypoxic CP/R using H9c2 cardiomyoblasts (unpublished observations).…”

“…Importantly, Bednarczyk et al showed in astrocytoma cells that BKCa channel subunits associate with mitochondrial ETC complexes and that enhanced mitochondrial respiration inhibits BKCa channel opening. 46 These findings raise the interesting possibility that BKCa channels promote increased ETC activity and are in turn negatively regulated by the ETC. Generally, enhanced respiration is thought to significantly contribute to enhanced ROS generation.…”