CANINE ROD PHOTORECEPTOR CGMP-GATED CHANNEL PROTEIN ALPHA-SUBUNIT - STUDIES ON THE EXPRESSION OF THE GENE AND CHARACTERIZATION OF THE CDNA

Rod photoreceptor cyclic GMP gated-channel protein is a key component of the visual transduction cascade in the vertebrate retina, The prote in is composed of at least two subunits (alpha and beta). Mutations in the alpha-subunit (CNGC1) have been shown to cause retinitis pigmento sa (RP) in humans. Several heterogeneous canine retinal diseases, whic h are clinically similar to RP, are known collectively as progressive retinal atrophy (PRA) and occur in dogs in a breed-specific manner. Fo r the purpose of examining CNGC1 gene as a candidate for PRA, we repor t here the characterization of canine CNGC1 cDNA, and examine the expr ession of the gene in different tissues by northern analysis, reverse transcription and polymerase chain reaction (RT-PCR), and retinal immu nocytochemistry. 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 t o encode a protein containing 691 amino acids which include six putati ve transmembrane domains, a pore loop and a cGMP binding domain as wel l as one potential extracellular site for N-linked glycosylation. Over the coding region, the canine CNGC1 shares 85-90% identity in the nuc leotide sequence and 91-94% identity in the deduced amino acid sequenc e with its homologues in other mammalian species. However, the homolog y drops to only 71% and 78% of shared nucleotide and predicted amino a cid 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, anothe r minor transcript (9.8 kb) is consistently observed in the canine ret inal RNA which may represent the canine homologue of the rod specific beta-subunit of the cyclic nucleotide-gated channel protein, Transcrip ts 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 . (C) 1997 Academic Press Limited.