Identification of the minimal promoter for specific expression of the GABA1 receptor in retinal bipolar cells

Abstract

γ-aminobutyric acid (GABA) receptors regulate rapid synaptic ion currents in the axon end of retinal ON bipolar neurons, acting as a point of control along the visual pathway. In the GABA1 subunit knock out mouse, inhibition mediated by this receptor is totally eliminated, showing its role in neural transmission in retina. GABA1 mRNA is expressed in mouse retina after post-natal day 7, but little is known about its transcriptional regulation. To identify the GABA1 promoter, in silico analyses were performed and indicated that a 0.290-kb fragment, flanking the 5'-end of the GABA1 gene, includes putative transcription factor-binding sites, two Inr elements, and lacks a TATA-box. A rapid amplification of cDNA ends (RACE) assay showed three transcription start sites (TSS) clustered in the first exon. Luciferase reporter assays indicated that a 0.232-kb fragment upstream from the ATG is the minimal promoter in transfected cell lines and in vitro electroporated retinae. The second Inr and AP1 site are important to activate transcription in secretin tumor cells (STC-1) and retina. Finally, the 0.232-kb fragment drives green fluorescent protein (GFP) expression to the inner nuclear layer, where bipolar cells are present. This first work paves the way for further studies of molecular elements that control GABA1 transcription and regulate its expression during retinal development. GABA receptors play a central role regulating rapid synaptic transmission in retinal bipolar neurons. Expression of GABA1 gene is timely controlled during early post-natal development, being transcriptionally active 7 days after birth in mice. A 232-bp fragment, flanking the 5'- end of the GABA1 gene, suffices to drive transcription in the inner nuclear layer of the retina. © 2012 International Society for Neurochemistry.