Isolation and characterization of a skate retinal GABA transporter cDNA.

PURPOSE

The inhibitory neurotransmitter gamma-aminobutyric acid (GABA) is believed to play a crucial role in the processing of information within the vertebrate retina. Extracellular concentrations of GABA are thought to be tightly regulated by carrier-mediated transport proteins in neurons and glial cells. The purpose of this work was to isolate the gene that encodes one of these transport proteins in the skate retina.

METHODS

cDNA clones were isolated from a skate retinal cDNA library using a mouse retinal GABA transporter (GAT1) cDNA as a probe. The PCR technique was used to fill sequence gaps, and 5' and 3' RACE were employed to amplify the 5' and 3' untranslated regions. The amplified fragments were subcloned into a T-vector. Blots containing RNA from 10 different tissues were probed to determine the size of the transcript and the tissue distribution.

RESULTS

Sequence analysis revealed that the skate retinal GABA transporter cDNA shared 72% identity with the mouse GABA transporter-1 at the DNA level and 80% identity at the amino acid level. Multiple sequence alignments showed that our sequence is closest to the Torpedo GABA transporter-1. Two transcripts, 4.5 and 7 kb, were detected in retina and possibly brain by RNA blot analysis. Fourteen introns were detected in the skate GABA transporter gene.

CONCLUSIONS

We successfully isolated a full length GABA transporter cDNA from the retina of the skate. The size of the full length sequence of the skate retinal GABA transporter is in agreement with the size of the smaller transcript detected on RNA blots. The larger transcript observed on the RNA blot may be the result of either alternative splicing or utilization of a downstream poly A signal.