title: Putative Structural and Functional Coupling of the Mitochondrial BKCa Channel to the Respiratory Chain.
abstract: Potassium channels have been found in the inner mitochondrial membranes of various cells. These channels regulate the mitochondrial membrane potential, the matrix volume and respiration. The activation of these channels is cytoprotective. In our study, the single-channel activity of a large-conductance Ca(2+)-regulated potassium channel (mitoBKCa channel) was measured by patch-clamping mitoplasts isolated from the human astrocytoma (glioblastoma) U-87 MG cell line. A potassium-selective current was recorded with a mean conductance of 290 pS in symmetrical 150 mM KCl solution. The channel was activated by Ca(2+) at micromolar concentrations and by the potassium channel opener NS1619. The channel was inhibited by paxilline and iberiotoxin, known inhibitors of BKCa channels. Western blot analysis, immuno-gold electron microscopy, high-resolution immunofluorescence assays and polymerase chain reaction demonstrated the presence of the BKCa channel β4 subunit in the inner mitochondrial membrane of the human astrocytoma cells. We showed that substrates of the respiratory chain, such as NADH, succinate, and glutamate/malate, decrease the activity of the channel at positive voltages. This effect was abolished by rotenone, antimycin and cyanide, inhibitors of the respiratory chain. The putative interaction of the β4 subunit of mitoBKCa with cytochrome c oxidase was demonstrated using blue native electrophoresis. Our findings indicate possible structural and functional coupling of the mitoBKCa channel with the mitochondrial respiratory chain in human astrocytoma U-87 MG cells.
authors.affiliation: Laboratory of Intracellular Ion Channels, Nencki Institute of Experimental Biology, Warsaw, Poland ; Department of Biophysics, Warsaw University of Life Sciences - SGGW, Warsaw, Poland.
authors.forename: Piotr
authors.initials: P
authors.lastname: Bednarczyk
authors.forename: Mariusz R
authors.initials: MR
authors.lastname: Wieckowski
authors.forename: Malgorzata
authors.initials: M
authors.lastname: Broszkiewicz
authors.forename: Krzysztof
authors.initials: K
authors.lastname: Skowronek
authors.forename: Detlef
authors.initials: D
authors.lastname: Siemen
authors.forename: Adam
authors.initials: A
authors.lastname: Szewczyk
mesh_terms: D000818:Animals; D001254:Astrocytoma / Q000378:metabolism / Q000473:pathology; D002118:Calcium / Q000378:metabolism; D002412:Cations / Q000378:metabolism; D045744:Cell Line, Tumor; D004579:Electron Transport / Q000187:drug effects / Q000502:physiology*; D003576:Electron Transport Complex IV / Q000378:metabolism; D005904:Glial Fibrillary Acidic Protein / Q000378:metabolism; D006801:Humans; D051036:Large-Conductance Calcium-Activated Potassium Channels / Q000378:metabolism*; D008564:Membrane Potentials / Q000187:drug effects / Q000502:physiology; D008928:Mitochondria / Q000187:drug effects / Q000378:metabolism; D011188:Potassium / Q000378:metabolism; D051381:Rats
chemical_list: D002412:Cations; D005904:Glial Fibrillary Acidic Protein; D051036:Large-Conductance Calcium-Activated Potassium Channels; D003576:Electron Transport Complex IV; D011188:Potassium; D002118:Calcium