IMMUNOLABELING OF THE NA+ CL--DEPENDENT ORPHAN TRANSPORTER RXT1 IN THE RAT CENTRAL-NERVOUS-SYSTEM/

Previous studies have shown that the mRNA encoding the Na+/Cl--depende nt ''orphan'' transporter Rxt1 is expressed exclusively in the central nervous system (CNS). In the present study, specific antibodies were raised in rabbits for the detailed mapping of this transporter in the rat. The C-terminal part of Rxt1 was fused with glutathione-S-transfer ase (Rxt1(ct)- GST) and the resulting fusion protein was used as antig en. The specificity of the antiserum toward Rxt1 was confirmed by immu nofluorescent, Western blot, and immunoautoradiographic experiments. I n cerebral cortex membranes, Rxt1-like material recognized by the anti serum is a glycosylated protein of 97-116 kDa. This protein was the mo st abundant in the caudate-putamen, followed, in decreasing order, by the cerebral cortex approximate to hippocampus > cerebellum > brainste m > spinal cord. In contrast, no immuno-reactive material could be det ected in peripheral tissues (tongue, thymus, heart, lung, spleen, kidn ey, adrenals, liver, skeletal muscle, intestine, testis). Immunoautora diographic labeling with affinity-purified anti-Rxt1(ct)-GST antibodie s showed high levels of Rxt1-like material in the olfactory bulb, cere bral cortex, striatal complex, hippocampal formation, superior layer o f the anterior colliculus, cortex, and deep nuclei in the cerebellum. The regional distribution of Rxt1-like material generally matched that of GABAergic and glutamatergic projections in agreement with previous in situ hybridization data. (C) 1995 Wiley-Liss, Inc.