Identification of a voltage-gated potassium channel in gerbil hippocampal mitochondria

Bednarczyk, Piotr; Kowalczyk, Joanna E.; Beresewicz, M; Dołowy, Krzysztof; Szewczyk, Adam; Zabłocka, Barbara

doi:10.1016/j.bbrc.2010.06.011

Cited by 54 publications

(34 citation statements)

References 33 publications

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“…Dual localization of the channel, i.e. expression of Kv1.3 in mitochondrial and plasma membranes, had already been found in breast cancer MCF-7, prostate cancer PC3, osteosarcoma SAOS-2, melanoma B16F10, Jurkat T cell lymphoma [22,26,27] and pancreas carcinoma [28]. In addition, recent data indicate the presence of Kv1.3 also in cis-Golgi [29] and in the nuclear membrane [30], which is also consistent with the present studies.…”

Section: Discussionsupporting

confidence: 80%

Targeting the Potassium Channel Kv1.3 Kills Glioblastoma Cells

et al. 2017

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Background/Aims: Glioblastoma (GBM) is one of the most aggressive cancers, counting for a high number of the newly diagnosed patients with central nervous system (CNS) cancers in the United States and Europe. Major features of GBM include aggressive and invasive growth as well as a high resistance to treatment. Kv1.3, a potassium channel of the shaker family, is expressed in the inner mitochondrial membrane of many cancer cells. Inhibition of mitochondrial Kv1.3 was shown to induce apoptosis in several tumor cells at doses that were not lethal for normal cells. Methods: We investigated the expression of Kv1.3 in different glioma cell lines by immunocytochemistry, western blotting and electron microscopy and analyzed the effect of newly synthesized, mitochondria-targeted, Kv1.3 inhibitors on the induction of cell death in these cells. Finally, we performed in vivo studies on glioma bearing mice. Results: Here, we report that Kv1.3 is expressed in mitochondria of human and murine GL261, A172 and LN308 glioma cells. Treatment with the novel Kv1.3 inhibitors PAPTP or PCARBTP as well as with clofazimine induced massive cell death in glioma cells, while Psora-4 and PAP-1 were almost without effect. However, in vivo experiments revealed that the drugs had no effect on orthotopic brain tumors in vivo. Conclusion: These data serve as proof of principle that Kv1.3 inhibitors kills GBM cells, but drugs that act in vivo against glioblastoma must be developed to translate these findings in vivo.

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Section: Discussionsupporting

confidence: 80%

Targeting the Potassium Channel Kv1.3 Kills Glioblastoma Cells

et al. 2017

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“…Patch-clamp experiments on endothelial mitoplasts were performed as previously described for mitoplasts obtained from other mammalian mitochondria (3,8). The experiments were carried out in mitoplast-attached single-channel mode using a pipette of borosilicate glass (Harvard, UK) with a mean resistance of ϳ15 M⍀, which was pulled using a Flaming/Brown Micropipette Puller.…”

Section: Methodsmentioning

confidence: 99%

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

Bednarczyk

Kozieł

Jarmuszkiewicz

et al. 2013

American Journal of Physiology-Heart and Circulatory Physiology

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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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“…28 In various systems, open potassium channels seem to keep the PTP closed. 29 Kv1.3 was recently found in mitochondria of prostate cancer and breast cancer cell lines PC3 and MCF-7, 24 in hippocampal neurons, 30 and in astrocytes. 29 The observation that the ring of negatively charged amino acids in the pore of potassium channels is structurally highly conserved in all voltage-gated potassium channels (see, e.g., Rauer et al 31 ) suggests that the action of Bax may not be limited to Kv1.3.…”

Section: Discussionmentioning

confidence: 90%

Single-point mutations of a lysine residue change function of Bax and Bcl-xL expressed in Bax- and Bak-less mouse embryonic fibroblasts: novel insights into the molecular mechanisms of Bax-induced apoptosis

et al. 2010

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Members of the Bcl-2 family play key roles as proapoptotic (e.g., Bax) and antiapoptotic (e.g., Bcl-x L ) regulators of programmed cell death. We previously identified the mitochondrial potassium channel Kv1.3 as a novel target of Bax. Incubating Kv1.3-positive isolated mitochondria with Bax triggered apoptotic events, whereas Kv1.3-deficient mitochondria were resistant to this stimulus. Mutation of Bax at lysine 128 (BaxK128E) abrogated its effects on Kv1.3 and the induction of apoptotic changes in mitochondria. These data indicate a toxin-like action of Bax on Kv1.3 to trigger at least some of the mitochondrial changes typical for apoptosis. To gain insight into the mechanism of Bax-Kv1.3 interaction, we mutated Glu158 of Bcl-x L (corresponding to K128 in Bax) to lysine. This substitution turned Bcl-x L proapoptotic. Transfection of double knockout (Bax À/À /Bak À/À ) mouse embryonic fibroblasts (DKO MEFs) with either wild-type Bax, BaxK128E, or Bcl-x L E158K showed that apoptosis induced by various stimuli was defective in DKO MEFs and BaxK128E-transfected cells, but was recovered upon transfection with Bcl-xLE158K or wild-type Bax. Both wild-type Bax and BaxK128E can form similar ion-conducting pores upon incorporation into planar lipid bilayers. Our results point to a physiologically relevant interaction of Bax with Kv1.3 and further indicate a crucial role of a distinct lysine in determining the proapoptotic character of Bcl2-family proteins.

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Identification of a voltage-gated potassium channel in gerbil hippocampal mitochondria

Cited by 54 publications

References 33 publications

Targeting the Potassium Channel Kv1.3 Kills Glioblastoma Cells

Targeting the Potassium Channel Kv1.3 Kills Glioblastoma Cells

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

Single-point mutations of a lysine residue change function of Bax and Bcl-xL expressed in Bax- and Bak-less mouse embryonic fibroblasts: novel insights into the molecular mechanisms of Bax-induced apoptosis

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Identification of a voltage-gated potassium channel in gerbil hippocampal mitochondria

Cited by 54 publications

References 33 publications

Targeting the Potassium Channel Kv1.3 Kills Glioblastoma Cells

Targeting the Potassium Channel Kv1.3 Kills Glioblastoma Cells

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

Single-point mutations of a lysine residue change function of Bax and Bcl-xL expressed in Bax- and Bak-less mouse embryonic fibroblasts: novel insights into the molecular mechanisms of Bax-induced apoptosis

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