Neuropharmacology 38 (1999) 785 – 792 Functional integrity of green fluorescent protein conjugated glycine receptor channels Brigitte David-Watine *, Spencer L. Shorte, Sergio Fucile, Didier de Saint Jan, Henri Korn, Piotr Bregestovski INSERM U-261, Institut Pasteur, 25 Rue du Dr. Roux, 75724 Paris Cedex 15, France Accepted 27 January 1999 Abstract The alpha subunit (Z1) of the zebrafish glycine receptor (GlyR) has been N-terminus fused with green fluorescent protein (GFP). We found that both pharmacological and electrophysiological properties of this chimeric Z1-GFP are indistinguishable from those of the wild-type receptor when expressed in Xenopus oocytes and cell lines. The apparent affinities of this receptor for agonists (glycine, taurine and GABA), and the antagonist (strychnine) are unchanged, and single channel kinetics are not altered. In the same expression systems, Z1-GFP was visualized using fluorescence microscopy. Fluorescence was distributed anisotrop- ically across cellular membranes. In addition to the Golgi apparatus and endoplasmic reticulum, its presence was also detected on the plasmalemma, localized at discrete hot -spots which were identified as sites of high membrane turnover. Overall, the preservation in Z1-GFPs of the wild type receptor functional properties makes it a promising new tool for further in situ investigations of GlyR expression, distribution and function. © 1999 Elsevier Science Ltd. All rights reserved. Keywords: Glycine; GFP; Imaging; Receptor sorting; Zebrafish; Ion channel 1. Introduction Glycinergic neurotransmission is important in the mechanisms of voluntary motor control, sensory signal- ing (Rajendra et al., 1997) and neocortical development (Flint et al., 1998). In the spinal cord and brain stem of higher (Rajendra et al., 1997) and lower vertebrates, e.g. the teleosts such as goldfish (Carassius auratus ) (Korn et al., 1990) and zebrafish (Danio rerio ) (Hatta and Korn, 1998) fast inhibitory neurotransmission is largely mediated via post-synaptic glycine receptors (GlyR). The heteromeric GlyR comprises  and  subunits in a 3:2 ratio (Langosch et al., 1988). However, recent reports have provided evidence for functional  ho- momeric channel formation in heterologous expression systems (Schmieden et al., 1989; Grenningloh et al., 1990) and in vivo (Legendre, 1997; Elster et al., 1998). Furthermore it has been suggested that specific target- ing of GlyR subunits is an important factor accounting for the functional responsiveness of the inhibitory post- synaptic membrane (Craig, 1998; Kirsch and Betz, 1998; Levi et al., 1998). Thus it is of critical interest to obtain an adequate description of the composition and distribution of GlyR subunits inside neurons in order to resolve the mechanisms underlying GlyR presentation at synapses. One experimental approach to achieve this goal is to visualize GlyRs directly in situ (Marshall et al., 1995; Ziff, 1997) in living tissue, an approach so far limited to fixed tissue (Triller et al., 1985). We have chosen the zebrafish as a model to investi- gate these questions. In addition to its glycinergic in- hibitory circuitry which controls well defined motor behavior (Hatta and Korn, 1998), this animal is a well established model for developmental studies (Zebrafish issue, 1996) and has already proved accessible to molec- ular strategies generating mutant strains for studies focused on motor diseases (Granato et al., 1996). The GlyR  subunit of zebrafish Z1 displays striking struc- tural and functional similarities with the human, murine and rat  subunits (David-Watine et al., 1999). * Corresponding author. Tel.: +33-1-45688876; fax: +33-1- 45688790. E-mail address: bdwati@pasteur.fr (B. David-Watine) 0028-3908/99/$ - see front matter © 1999 Elsevier Science Ltd. All rights reserved. PII:S0028-3908(99)00015-5