RETINA-specific expression of kcnv2 is controlled by cone-rod homeobox (Crx) and neural retina leucine zipper (Nrl)

Abstract

Cone dystrophy with supernormal rod response (CDSRR) is an autosomal recessive disorder that leads to progressive retinal degeneration with a distinct electroretinogram (ERG) phenotype. CDSRR patients show reduced sensitivity to dim light, augmented response to suprathreshold light and reduced response to flicker. The disorder is caused by mutations in the KCNV2 gene, which encodes the Kv11.1 subunit of a voltage-gated potassium channel. Here, we studied the retina- specific expression and cis-regulatory activity of the murine Kcnv2 gene using electroporation of explanted retinas. Using qRT-PCR profiling of early postnatal retinas, we showed that Kcnv2 expression increased towards P14, which marks the beginning of visual activity in mice. In vivo electroporation of GFP-Kcnv2 expressing plasmids revealed that Kv11.1 localizes to the inner segment membranes of adult P21 photoreceptors. Using bioinformatic prediction and chromatin immunoprecipitation (ChIP), we identified two Crx binding sites (CBS) and one Nrl binding site (NBS) in the Kcnv2 promoter. Reporter electroporation of the wild type promoter region induced strong DsRed expression, indicating high regulatory activity, whereas shRNA-mediated knockdown of Crx and Nrl resulted in reduced Kcnv2 promoter activity and low endogenous Kcnv2 mRNA expression in the retina. Sitedirected mutagenesis of the CBS and NBS demonstrated that CBS2 is crucial for Kcnv2 promoter activity. We conclude that nucleotide changes in evolutionary conserved CBS could impact retina-specific expression levels of Kcnv2.