ION CHANNELS, RECEPTORS AND TRANSPORTERS Identification and characterization of the zebrafish ClC-2 chloride channel orthologs Carla Pérez-Rius & Héctor Gaitán-Peñas & Raúl Estévez & Alejandro Barrallo-Gimeno Received: 30 July 2014 /Revised: 9 September 2014 /Accepted: 9 September 2014 # Springer-Verlag Berlin Heidelberg 2014 Abstract ClC-2 is a Cl - channel that belongs to the CLC family of chloride channel/transport proteins. ClC-2 molecu- lar role is not clear, and Clcn2 knockout mice develop blind- ness, sterility, and leukodystrophy by unknown reasons. ClC- 2 is associated in the brain with the adhesion molecule GlialCAM, which is defective in a type of leukodystrophy, involving ClC-2 in the homeostasis of myelin. To get more insight into the functions of ClC-2, we have identified in this work the three ClC-2 orthologs in zebrafish. clcn2a and clcn2b resulted from the teleost-specific whole genome dupli- cation, while clcn2c arose from a gene duplication from clcn2b. The expression patterns in adult tissues and embryos of zebrafish clcn2 paralogs support their subfunctionalization after the duplications, with clcn2a being enriched in excitable tissues and clcn2c in ionocytes. All three zebrafish clc-2 proteins interact with human GLIALCAM, that is able to target them to cell junctions, as it does with mammalian ClC-2. We could detect clc-2a and clc-2b inward rectified chloride currents with different voltage-dependence and ki- netics in Xenopus oocytes, while clc-2c remained inactive. Interestingly, GlialCAM proteins did not modify clc-2b in- ward rectification. Then, our work extends the repertoire of ClC-2 proteins and provides new tools for structure-function and physiology studies. Keywords ClC-2 Cl - channel . In situ hybridization . Xenopus oocyte . Genomeduplication . Zebrafish . GlialCAM Introduction Chloride is the main anion with biological functions. It is involved in a plethora of roles, such as cell volume regulation, transepithelial fluid movement, muscle contraction, charge compensation, and acidification of intracellular organelles. Cellular chloride levels are actively regulated by different co-transporters, exchangers, and channels [7]. Among them, ClC proteins, which are found in all phyla, in vertebrates constitute a family of proteins divided in two subgroups: one group of intracellular Cl - /H + exchangers with five members, and another group of Cl - channels located at the plasma membrane [22]. This latter group is formed by four members in mammals: ClC-1, expressed in the skeletal muscle and involved in myotonia congenita [29, 48, 49]; ClC-Ka and ClC-Kb, expressed in renal tubules and the inner ear, respon- sible for some variants of Bartter’ s syndrome (reviewed in [13]); and ClC-2, broadly expressed in many tissues [51], that has been involved in a leukodystrophy which develops with intramyelinic oedema [6]. ClC proteins function as homodimers [36, 38], and every subunit contains one ion pore [8]. These two ion pores operate independently of each other, but there is a common gating mechanism which affects both pores simultaneously, as first deduced by Miller [39]. Heterologous expression of heterodi- meric chloride channels has shown that there are intersubunit interactions that affect individual and common gating [35, 50, 54]. ClC-2 is activated by membrane hyperpolarization, ex- tracellular acidic pH, cell swelling, and an increase in CPR and HGP contributed equally to this work. Electronic supplementary material The online version of this article (doi:10.1007/s00424-014-1614-z) contains supplementary material, which is available to authorized users. C. Pérez-Rius : H. Gaitán-Peñas : R. Estévez (*) : A. Barrallo-Gimeno (*) Departament de Ciències Fisiològiques II, Unitat de Fisiologia, Universitat de Barcelona, Carrer Feixa Llarga s/n, L´Hospitalet de Llobregat 08907, Barcelona, Spain e-mail: restevez@ub.edu e-mail: abarrallo@ub.edu R. Estévez : A. Barrallo-Gimeno U750, Centro de Investigación Biológica en Red de Enfermedades Raras (CIBERER), Instituto de Salud Carlos III, Barcelona, Spain Pflugers Arch - Eur J Physiol DOI 10.1007/s00424-014-1614-z