TMC8 (EVER2) attenuates intracellular signaling by Zn 2+ and Ca 2+ and suppresses activation of Cl - currents Lalida Sirianant, Jiraporn Ousingsawat, Yuemin Tian, Rainer Schreiber, Karl Kunzelmann ⁎ Institut für Physiologie, Universität Regensburg, Universitätsstraße 31, D-93053 Regensburg, Germany abstract article info Article history: Received 19 August 2014 Received in revised form 1 September 2014 Accepted 2 September 2014 Available online 15 September 2014 Keywords: Transmembrane channel-like protein TMC8 EVER2 Zn 2+ signaling LRRC8A Anoctamin 1 Eight paralogue members form the family of transmembrane channel-like (TMC) proteins that share consider- able sequence homology to anoctamin 1 (Ano1, TMEM16A). Ano1 is a Ca 2+ activated Cl - channel that is related to head and neck cancer, often caused by human papilloma virus (HPV) infection. Mutations in TMC 6 and 8 (EVER1, EVER2) cause epidermodysplasia verruciformis. This rare skin disease is characterized by abnormal sus- ceptibility to HPV infection and cancer. We found that in contrast to Ano1 the common paralogues TMC4–TMC8 did not produce Ca 2+ activated Cl - currents when expressed in HEK293 cells. On the contrary, TMC8 was found to be localized in the endoplasmic reticulum (ER), where it inhibited receptor mediated Ca 2+ release, activation of Ano1 and volume regulated LRRC8-related Cl - currents. Zn 2+ is co-released from the ER together with Ca 2+ and thereby further augments Ca 2+ store release. Because TMC8 is required to lower cytosolic Zn 2+ concentra- tions by the Zn 2+ transporter ZnT-1, we hypothesize that HPV infections and cancer caused by mutations in TMC8 are related to upregulated Zn 2+ /Ca 2+ signaling and activation of Ano1. © 2014 Elsevier Inc. All rights reserved. 1. Introduction The transmembrane channel-like (TMC) family of proteins consists of 8 members in mammals. These proteins have been implicated in human diseases like hearing loss and human papillomavirus (HPV) in- fections, leading to epidermodysplasia verruciformis and cancer [1–3]. There is a noticeable overlap between TMC proteins and anoctamins, which suggested that these proteins may form a super family [4]. Ten paralogue proteins form the anoctamin family of proteins (Ano1-10, TMEM16A-K). It was shown for Ano1, 2, and 6 that they can produce Ca 2+ activated Cl - channels [5–11], while Ano6 and other anoctamins have additional functions [9,12–14]. Overexpression of anoctamin 1 is observed in head and neck cancer (HNSCC) and other tumors, where they support proliferation and tumor development [15–19]. Notably, about half of all HNSCC are caused by infection with HPV [20–22]. TMC4–7 but not TMC8 show relatively broad expression [3]. Individ- uals with mutations in TMC6 or TMC8 (EVER1, EVER2) demonstrate high susceptibility towards infections with HPV, which turns into uncon- trolled HPV infections, growth of scaly macules and papules, with a high risk of turning into cancer [23,24]. It was shown that TMC6 and TMC8 activate the ER-located zinc transporter ZnT-1, thereby facilitating Zn 2+ uptake into the endoplasmic reticulum, a mechanism that appears essential to protect from HPV-infections. Mutations in TMC6 and TMC8 may therefore lead to enhanced cytosolic Zn 2+ levels with leakage of Zn 2+ into the nucleus and enhanced transcription of Zn 2+ -dependent viral proteins [24,25]. Noticeable similarities exist between HNSCC and epidermodysplasia verruciformis, which suggest common pathogenic pathways [26]. Because of the potential structural similarities between anoctamins and TMC proteins, we asked whether TMC proteins are able to produce Ca 2+ activated Cl - currents. However, while activation of Cl - currents was not observed, we found for TMC8 suppression of Ca 2+ and volume activated Cl - currents. The suppression of Ano1 currents was due to the attenuation of intracellular Ca 2+ signaling, which was controlled by cy- tosolic Zn 2+ levels. The data demonstrate for the first time that TMC8 limits activation of Ca 2+ -dependent and volume activated whole cell currents, probably by controlling compartmentalized intracellular Ca 2+ signaling. 2. Experimental procedures 2.1. Cell culture, cloning of TMCs, siRNA HEK293, Fisher rat thyroid (FRT), HT 29 , and Cal33 cells were grown in DMEM-F12 and OptiMEM medium, respectively. All media were supplemented with 10% FBS. Cells were incubated in 5% CO 2 at 37 °C. Plasmids were transfected into cells using standard methods (Lipofectamine, Invitrogen). Cells were examined 48 or 72 h after transfection. cDNA of TMC4 (IRAUp969G1081D); TMC5 (IRAKp961O07168Q), TMC6 (IRAUp969E0379D), TMC7 (IRATp970H0474D) and TMC8 (IRATp970F12109D) (ImaGenes; Berlin, Germany) were subcloned into pcDNA31. Fusion proteins with EGFP were generated by PCR techniques. All cDNAs were Cellular Signalling 26 (2014) 2826–2833 ⁎ Corresponding author. Tel.: +49 941 943 4302; fax: +49 941 943 4315. E-mail address: karl.kunzelmann@ur.de (K. Kunzelmann). http://dx.doi.org/10.1016/j.cellsig.2014.09.001 0898-6568/© 2014 Elsevier Inc. All rights reserved. Contents lists available at ScienceDirect Cellular Signalling journal homepage: www.elsevier.com/locate/cellsig