259 Efects of Cd 2+ on the epithelial Na + channel (ENaC) investigated by experimental and modeling studies Maria Mernea 1 , Roxana Ulaˇreanu 1 , Octavian Călborean 1 , Sergiu Chira 2 , Octavian Popescu 2 , Dan F. Mihailescu 1 and Dana Cucu 1 1 Department of Anatomy, Physiology and Biophysics, Faculty of Biology, University of Bucharest, Bucharest, Romania 2 Molecular Biology Centre, Institute for Interdisciplinary Experimental Research, University “Babes-Bolyai”, 42 August Treboniu Laurean, 400271 Cluj-Napoca, Romania Abstract. Te function of the epithelial Na + channel from the apical membrane of many Na + trans- porting epithelia is modulated by various chemical compounds from the extracellular space, such as heavy metals, protons or chloride ions. We have studied the efect of extracellular Cd 2+ on the function of the epithelial Na + channel (ENaC) in heterologously expressed Xenopus laevis oocytes and Na + -transporting epithelia. We assayed channel function as the amiloride-sensitive sodium cur- rent (I Na ). Cd 2+ rapidly and voltage-independently inhibited I Na in oocytes expressing αβγ Xenopus ENaC (xENaC). Te extracellular Cd 2+ inhibited Na + transport and showed no infuence on ENaC trafcking, as revealed by concomitant measurements of the transepithelial current, conductance and capacitance in Na + -transporting epithelia. Instead, amiloride inhibition was noticeably diminished in the presence of Cd 2+ on the apical membrane. Using molecular modeling approaches, we describe the amiloride binding sites in rat and xENaC structures, and we present four putative binding sites for Cd 2+ . Tese results indicate that ENaC functions as a sensor for external Cd 2+ . Key words: ENaC — Xenopus laevis — Cd 2+ — A6 epithelia Gen. Physiol. Biophys. (2016), 35, 259–271 doi: 10.4149/gpb_2015054 Correspondence to: Dana Cucu, Department of Anatomy, Physi- ology and Biophysics, Faculty of Biology, University of Bucharest, Bucharest, Romania E-mail: dana.cucu@bio.unibuc.ro Introduction Cadmium has been described as one of the most harm- ful pollutants since the 19th century, when exposure to cadmium was seen as a problem confned especially to occupational exposure. Today, it is widely accepted that the main source of cadmi- um is cigarette smoke. Tis source and the accumulation of the metal in tissues and organs are the concerns of the modern world. New data from the genomic era show that cadmium afects the human and animal genome with efects relevant to tumorigenesis, tumor development and many other countless insults to human and animal organs (reviewed in Tevenod et al. 2013). Te current theory suggests that cadmium has no direct genotoxic efects but rather a multitude of combined mechanisms leading to the dysregulation of cellular events such as cell growth, apoptosis and proliferation. From the start of toxicology studies, the kidney was identifed as the critical organ in cadmium exposure in hu- mans and animals, and acute cytotoxic efects were related to tubular damage (Nordberg et al. 2009). Tubular damage was indicated to follow from oxidative stress (Cucu et al. 2011), afecting the vectorial active transport (Bathula et al. 2008) or damaging the epithelial barrier (Boveri et al. 2004). Tese processes were associated with the impaired function of various renal transporters. For instance, the ionic form of cadmium blocks the currents through the epithelial Ca 2+ channel (ECaC), heterologously expressed in HEK cells (Vennekens et al. 2001), whereas cadmium- methallothionein-1 (Cd-MT) directly blocks the apical Na + - glucose cotransporter on the luminal side in the proximal tubule (Tsuruoka et al. 2008). In the proximal tubular brush border, Cd 2+ inhibits the transport of the inorganic sulfate Electronic supplementary material. Figures S1, S2 and S3. Te online version of this article (doi:10.4149/gpb_2015054) contains supplementary material, which is available to authorized users.