A NOVEL GLYCINE RECEPTOR Z1 SUBUNIT VARIANT IN THE ZEBRAFISH BRAIN V. DEVIGNOT, L. PRADO DE CARVALHO, P. BREGESTOVSKI AND C. GOBLET* Laboratoire de Biologie Cellulaire et Mole ´ culaire du Neurone, Institut National de la Sante ´ et de la Recherche Me ´ dicale U261, De ´ partement des Biotechnologies, Institut Pasteur, 25 rue du Dr Roux, F-75724, Paris, France Abstract— subunits of the inhibitory glycine receptor (GlyR) display genetic heterogeneity in mammals and zebrafish. This diversity is increased in mammals by the alternative splicing mechanism. We report here in zebrafish, the charac- terization of a new Z1 subunit likely arising from Z1 gene by an alternative splice process (Z1L). This novel cDNA possesses 45 supplementary nucleotides at the putative exon2/exon3 boundary. The corresponding protein contains 15 additional amino acids in the NH2-terminal domain. Heter- ologous expression of homomeric GlyRZ1L in human em- bryonic kidney-293 cells generates glycine-gated strychnine- sensitive chloride channels with no obvious discrepancy with pharmacological properties of GlyRZ1. Moreover, zinc modulation of glycine-induced currents is identical in Z1 and Z1L glycine receptors. During ontogenesis, simulta- neous Z1 and Z1L mRNA synthesis have been observed. Embryonic and adult Z1 and Z1L mRNA expressions are restricted to the CNS. Embryonic Z1L mRNA anatomical pattern of expression is, however, highly restrained and strictly limited to the rostral part of the brain revealing a highly regionalized function of Z1L in the CNS. This report contributes to the characterization of the diversity of glycine receptor isoforms in zebrafish and emphasizes the common mechanism used among vertebrates for creating GlyR variety and specificity. © 2003 IBRO. Published by Elsevier Ltd. All rights reserved. Key words: alternative splicing, heterologous expression, ionic currents, zinc modulation, developmental profile, hy- bridization in situ. Glycine is the major inhibitory neurotransmitter in the mammalian and teleostean spinal cord and brain stem. The receptors for glycine belong to the ligand-gated ionic channel superfamily (LGIC, Grenningloh et al., 1987; Or- tells and Lunt, 1995) and are conserved subunits consti- tuted of a large NH 2 terminal part, 4-transmembrane (TM) domains, a large variable intracytoplasmic loop and a short extracytoplasmic COOH domain. Glycinergic receptors are pentameric chloride channel protein-activated by glycine, taurine, and alanine, and inhibited by strychnine at nano- molar concentrations (Rajendra et al., 1997). In several mammalian species, defects in glycinergic neurotransmis- sion have been implicated in complex motor disorders characterized by hypertonia and an exaggerated starle reflex (for review, Rajendra et al., 1997; Breitinger and Becker, 1998). The glycine receptor exists in several de- velopmentally and regionally regulated isoforms in the CNS. Molecular cloning data have disclosed heterogeneity of GlyR subunits in mammals (Betz et al.,1999; Rajendra et al., 1997) and in zebrafish (David-Watine et al., 1999; Imboden et al., 2001a,b,c). In addition to this heterogenity resulting from different GlyR subunit genes, several spliced variants have been isolated in mammalian species: 1ins in rat (Malosio et al., 1991), mice (Ryan et al., 1994) and cow (Pierce et al., 2001), 2A and 2B in rat (Kuhse et al., 1991) and finally 3K and 3L in human (Nikolic et al., 1998). Presence of splice variants is a shared property in the LGIC superfamily because many splice variants have been reported for acetylcholine (Mihovilovic et al., 1993; Mileo et al., 1995), serotonin (Hope et al., 1993) and GABA A re- ceptors (Whiting et al., 1990; Kofuji et al., 1991; Martinez- Torres et al., 1998), creating a diversity for synaptic func- tion. This mechanism of alternative splicing greatly in- creases the molecular diversity, a feature of particular importance to the cellular diversity of the nervous system. We report here, in the teleost brain of zebrafish Danio Rerio, identification, pharmacological properties, develop- mental and anatomical characteristics of a novel glycine receptor 1 subunit, Z1L, originating from Z1 gene by a splicing phenomenon. EXPERIMENTAL PROCEDURES Animal care Commercially available 4-month-old zebrafish were maintained in a laboratory breeding colony at 28.5 °C on a 14-h light/10-h dark cycle. Embryos were collected after natural spawning and were reared at 28.5 °C in embryo medium. Embryos were staged ac- cording to Kimmel et al. (1995) and aged as hours post-fertilization (hpf). cDNAs isolation The reported clone has been isolated by 5'/3' rapid amplification of cDNA ends (RACE) polymerase chain reaction (PCR) (Frohman et al., 1988). The experiments have been performed on zebrafish *Corresponding author. Tel: +33-1-44-32-39-83; fax: +33-1-44-32- 39-88. E-mail address: goblet@wotan.ens.fr (C. Goblet). Abbreviations: DIG, digoxigenin; GFP, green fluorescent protein; GlyR, glycine receptor; HEK-293 cells, human embryonic kidney-293 cells; HEPES, N-(2-hydroxyethyl)piperazine-N'-(2-ethanesulfonic acid); INSERM, Institut National de la Sante ´ et de la Recherche Me ´ dicale; LGIC, ligand-gated ionic channel; hpf, hours post-fertilization; PCR, polymerase chain reaction; RACE, rapid am- plification of cDNA ends; RT-PCR, reverse-transcriptase polymerase chain reaction; TM, transmembrane; UTP, uridine triphosphate; UTR, untranslated region. Neuroscience 122 (2003) 449 – 457 0306-4522/03$30.00+0.00 © 2003 IBRO. Published by Elsevier Ltd. All rights reserved. doi:10.1016/S0306-4522(03)00171-4 449