Canine rod photoreceptor cGMP-gated channel protein alpha-subunit: studies on the expression of the gene and characterization of the cDNA

Abstract

Rod photoreceptor cyclic GMP gated-channel protein is a key component of the visual transduction cascade in the vertebrate retina. The protein is composed of at least two subunits (alpha and beta). Mutations in the alpha-subunit (CNGC1) have been shown to cause retinitis pigmentosa (RP) in humans. Several heterogeneous canine retinal diseases, which are clinically similar to RP, are known collectively as progressive retinal atrophy (PRA) and occur in dogs in a breed-specific manner. For the purpose of examining CNGC1 gene as a candidate for PRA, we report here the characterization of canine CNGC1 cDNA, and examine the expression of the gene in different tissues by northern analysis, reverse transcription and polymerase chain reaction (RT-PCR), and retinal immunocytochemistry. The characterized canine CNGC1 cDNA sequence contains 2717 nucleotides which include 211 bp 5"-untranslated region and 430 bp 3"-untranslated region including the poly A tail. It is predicted to encode a protein containing 691 amino acids which include six putative transmembrane domains, a pore loop and a cGMP binding domain as well as one potential extracellular site for N-linked glycosylation. Over the coding region, the canine CNGC1 shares 85-90% identity in the nucleotide sequence and 91-94% identity in the deduced amino acid sequence with its homologues in other mammalian species. However, the homology drops to only 71% and 78% of shared nucleotide and predicted amino acid sequences, respectively, when compared to the chicken CNGC1. Among all the tissues examined the gene is expressed at a much higher level in retina as a major transcript of 3.5 kb length. In addition, another minor transcript (9.8 kb) is consistently observed in the canine retinal RNA which may represent the canine homologue of the rod specific beta-subunit of the cyclic nucleotide-gated channel protein. Transcripts were detected only in retina by northern analysis but low level of expression of CNGC1 was detected in liver, kidney, heart and brain by RT-PCR. The expression of the CNGC1 protein was found to be localized specifically to the photoreceptor outer segment by immunocytochemistry.