Research Article A large-conductance calcium-regulated K + channel in human dermal fibroblast mitochondria Anna Kicinska 1, *, Bartlomiej Augustynek 2, *, Bogusz Kulawiak 2 , Wieslawa Jarmuszkiewicz 1 , Adam Szewczyk 2 and Piotr Bednarczyk 3 1 Laboratory of Bioenergetics, Adam Mickiewicz University, Poznan, Poland; 2 Laboratory of Intracellular Ion Channels, Nencki Institute of Experimental Biology, 3 Pasteur St., Warsaw 02-093, Poland; and 3 Department of Biophysics, Warsaw University of Life Sciences (SGGW), Warsaw, Poland Correspondence: Adam Szewczyk (adam@nencki.gov.pl) Potassium channels have been found in the inner mitochondrial membrane of various cells. These channels regulate the mitochondrial membrane potential, respiration and pro- duction of reactive oxygen species. In the present study, we identified the activity of a mitochondrial large-conductance Ca 2+ -regulated potassium channel (mitoBK Ca channel) in mitoplasts isolated from a primary human dermal fibroblast cell line. A potassium selective current was recorded with a mean conductance of 280 ± 2 pS in a symmetrical 150 mM KCl solution. The mitoBK Ca channel was activated by the Ca 2+ and by potas- sium channel opener NS1619. The channel activity was irreversibly inhibited by paxilline, a selective inhibitor of the BK Ca channels. In isolated fibroblast mitochondria NS1619 depolarized the mitochondrial membrane potential, stimulated nonphosphorylating respir- ation and decreased superoxide formation. Additionally, the α- and β-subunits ( predomin- antly the β3-form) of the BK Ca channels were identified in fibroblast mitochondria. Our findings indicate, for the first time, the presence of a large-conductance Ca 2+ -regulated potassium channel in the inner mitochondrial membrane of human dermal fibroblasts. Introduction Fibroblasts are a predominant cell type in the dermis and play a crucial role in the regulation of skin physiology and pathology. They produce and organize the extracellular matrix of the dermis and par- ticipate in the stimulation of cell proliferation and apoptosis, wound repair, immune responses and inflammatory processes because they can release multiple growth factors and cytokines [1,2]. Dermal fibroblasts have been previously used to study calcium dynamics [3], membrane composition [4] and redox homeostasis [5], and to investigate mitochondrial dysfunction [6]. Many recent studies have focused on mitochondrial function as an emerging important element of skin pathology [7,8]. Several skin disorders (e.g. Aicardi–Goutiéres syndrome, epidermolysis bullosa simplex with muscular dys- trophy, lipodystrophies and Rothmund–Thomson syndrome) are linked to alterations of mitochon- drial energy metabolism (for a review, see ref. [9]). Clear evidence indicates that ion channels present in the inner mitochondrial membrane have a profound effect on mitochondrial metabolism and the efficiency of oxidative phosphorylation [10]. Ion transport through the inner mitochondrial membrane contributes to the regulation of the volume of the mitochondrial matrix, modulates the tightness of the coupling between mitochondrial respir- ation and ATP synthesis, and affects the mitochondrial membrane potential (ΔΨ), calcium transport, reactive oxygen species (ROS) production, mitochondrial dynamics and mitophagy [11–13]. Furthermore, mitochondrial potassium channel activation has been observed to provide cytoprotection against ischemic damage in various mammalian cells [14,15]. A mitochondrial large-conductance Ca 2+ -regulated potassium channel (mitoBK Ca channel) has been found in the inner mitochondrial membrane of the human glioma cell line LN229 [16], skeletal muscle [17], brain [18–20], heart [21,22] and endothelial cells [23]. All of the mitochondrial channels *These authors contributed equally to this work. Accepted Manuscript online: 11 October 2016 Version of Record published: 25 November 2016 Received: 3 August 2016 Revised: 23 September 2016 Accepted: 10 October 2016 © 2016 The Author(s); published by Portland Press Limited on behalf of the Biochemical Society 4457 Biochemical Journal (2016) 473 4457–4471 DOI: 10.1042/BCJ20160732 Downloaded from https://portlandpress.com/biochemj/article-pdf/473/23/4457/737280/bcj-2016-0732.pdf by guest on 17 June 2020