Excitatory amino acid receptors (EAARs) are the site of action of different psychoactive drugs, and have been implicated in simple forms of learning and memory. A natural ligand of EAARs is glutamate, which activates three pharmacologically distinct sites known as the kainate, AMPA, and NMDA receptors. Kainate activates the opening of a channel selective for sodium and calcium. In collaboration with Dr. R. Wenthold's group, the properties of a cDNA coding for a frog kainic acid binding protein (KABP) were studied. Results obtained using a Xenopus oocyte expression system, in conjunction with two-electrode voltage clamp), showed that the transcripts coded by the frog KABP cDNA can not form functional channels in injected oocytes. The conclusion from these results are that the cDNA codes for a putative subunit of the kainate receptor that cannot form functional channels in absence of other structural components, or that it codes for a KABP with a function not presently understood. In mammals kainic acid receptors (KARS) are particularly abundant in the cerebellum; present at higher densities in the molecular and granular cell layers. We have chosen to study KAR expression in the cerebellum because the cell types, organization, and development of this area have been extensively studied. Oligonucleotides, corresponding to the sequence of a recently cloned rat kainate receptor subunit (KAR1), were used to screen a rat cerebellum cDNA library at low stringency. A novel 3.9 kb cDNA clone was obtained that spans the entire coding region; this clone was called KAR2. The deduced amino acid sequence of the KAR2 cDNA codes for a protein that is 60% homologous with KAR1. Most of the homology resides in the portion of the protein predicted to code for membrane spanning regions. Northern blot analysis was used to determine the regional distribution of KAR2 mRNAs. The KAR2 transcripts are more abundant in cerebellum than in hippocampus, cortex, thalamus, and spinal cord. Preliminary studies using cerebellar tissue cultures suggest that KAR2 transcripts are present in granule cells and absent in cerebellar astrocytes; cerebellar astrocytes have been shown to respond to kainate application. Experiments are in progress to study the expression of KAR2 during development. In the future we plan to study the molecular mechanisms underlying the cell-specific and developmental expression of the KAR2 gene, as well as, other genes coding for EAARs.
