European Journal zyxwvutsrq of Neuroscience, Vol. 8, pp. 127-137, zyxwvutsr 1996 0 European Neuroscience Association z Distribution and Sites of Synthesis of NTT4, an Orphan Member of the N a+/CI--dependent Neu rotransmitter Transporter Family, in the Rat CNS zyx J. M. Luque, F. Jursky', N. Nelson' and J. G. Richards Pharma Division, Preclinical Research, F. Hoffmann-La Roche Ltd, CH-4002 Basel, Switzerland 'Roche Institute of Molecular Biology, Nutley, NJ 07110, USA. zyxwvuts Keywords: carrier, glutamate/glycine, immunohistochemistry, in situ hybridization Abstract The distribution and sites of synthesis in rat CNS of NTT4, a novel orphan member of the Na+/CI--dependent neurotransmittertransporter family, were determined by immunohistochemistry and hybridization histochemistry. Antibodies raised against recombinant fusion proteins, corresponding to residues of NlT4, and 35S-labelled oligodeoxyribonucleotide probes, were used to delineate the cellular distribution of the transporter at the protein and mRNA levels. High levels of immunoreactivity (mainly in the neuropil) were found in the olfactory bulb, cerebral cortex, striatum, hippocampus, thalamus, substantia nigra, pontine nuclei, cerebellum and spinal cord. The lowest levels were associated with the lateral hypothalamic area and deep mesencephalic nuclei. In sifu hybridization signals correlated well with the immunoreactivity, and demonstrated a widespread distribution of NlT4 transcripts exclusively in neurons. NTT4 transcripts appeared widely codistributed with the N-methyl-D- aspartate receptor subunit 1 (1-4b), i.e. spliced variants characterized by a common 5' zyxw 63 bp insertion. These results indicate that the transporter was associated with neuronal processes in specific glutamate innervated CNS regions. Although the substrate transported by N l T 4 remains unknown, our findings suggest a possible role for this carrier protein in glutamate/glycine neurotransmission. Introduction High-affinity transporters inserted in the plasma membrane of neurons or surrounding glial cells at synapses mediate the removal of released neurotransmitters, thereby terminating their action. Such neuro- transmitter influx is coupled to transmembrane ion gradients that provide the energy for transport against a concentration gradient (e.g. Amara and Kuhar, 1993). To date almost 20 genes encoding membrane neurotransmitter transporters have been cloned and expressed. They appear to fall into two structurally and mechanistically distinct gene families: (i) a Naf-dependent excitatory amino acid transporter family (Kanai and Hediger, 1992; Stork et al., 1992; Pines et al., 1993) and (ii) a Na+/CI--dependent family which includes the transporters for y-aminobutyric acid (GABA) (Guastella et al., 1990; Nelson et al., 1990; Borden et al., 1992; Clark et al., 1992; Liu et zyxwvutsr al., 1992a. 1993a; Lopez-Corcuera et al., 1992), glycine (Guastella et al., 1992; Liu et al., 1992c, 1993b; Smith et al., 1992b; Borowsky et al., 1993; Kim et al., 1994), noradrenalin/adrenalin (Pacholczyk et al., 1991), dopamine (Giros et al., 1991, 1992; Kilty et al., 1991; Shimada et al., 1991; Usdin et al., 1991; Vandenbergh et al., 1992) and serotonin (Blakely et al., 1991; Hoffman et al., 1991), as well as those for proline (Fremeau et al., 1992), betaine (Yamauchi et al., 1992). taurine (Liu et al., 1992b; Smith et al., 1992a; Uchida et al., 1992) and creatine (Mayser et al., 1992; Gimbal and Kilimann, 1993; Schloss et al., 1994). Recently, a rat cDNA encoding a novel protein (presumably belonging to the Na+/CI--dependent neurotransmitter transporter family) was cloned and sequenced (Liu et al., 1993~; Mestikawy et al., 1994). The cDNA was identified as a transcript of the gene N7T4 (also called XTI), of which a partial genomic clone was previously sequenced (Liu et al., 1992a). The NTT4 gene encodes 727 amino acids (Liu et al., 1993~). which is -100 residues larger than the average size (-600 amino acids) of other identified members of the Na+/CI--dependent transporter family. The analysis revealed a deviation from the conserved structure of most members of the family (35-38% sequence identity). Efforts to identify the specific substrate transported by " l T 4 in transiently transfected and stable cell lines have failed (Liu et al., 1993~; Mestikawy et al., 1994). Structurally, the protein encoded by this gene is similar (-60% sequence identity) to other published clones of this same gene family (eg V7-3-2) whose proteins have been termed 'orphan' transporters because their substrates are still unknown (Uhl et al., 1992). The specific function of such proteins might be revealed by mapping their distribution as well as their sites of synthesis in the CNS, to provide a putative association with known neurotransmitter or modulator pathways. In the present study, antibodies raised against recombinant fusion proteins, corresponding to residues of "IT4, were used to delineate Correspondence ro: Dr Juan M. Luque, Biocenter of the University Basle, Department of Pharmacology, Klingelbergstrasse 70, CH-4056 Basle, Switzerland Received 3 April zyxwvutsrqpon 1995. revised 8 June 1995. accepted 18 August 1995