Monitoring CNGA3 and CNGB3 Subunit Expression in Retinas of Day-Blind Canines with Inherited Deletion or Missense CNGB3 Asp262Asn Mutations Show Progressive Loss of Both CNGB3 and CNGA3 Expression

Abstract

Cone cyclic nucleotide-gated (CNG) channels are heteromeric, composed of CNGA3 and CNGB3 subunits. Inherited canine day blindness is similar to human achromatopsia and results in loss of cone function. Affected dogs have inherited deletion (-/-) or missense (m/m) Asp262Asn mutations in the CNGB3 gene. Cone ERG studies from m/m dogs show early, progressive loss of cone function with complete loss by ∼6-weeks. Immunohistochemical analysis of m/m and -/- retinal tissues using an anti-CNGA3 antibody show outer segment expression until ∼6 weeks; no immunoreactivity is observed at 1 year. A polyclonal antibody was generated against the C-terminus of canine CNGB3 to investigate expression in the m/m dogs. Immunohistochemistry on 6-week, 8-week and one-year old m/m retinas showed no expression of CNGB3. Immunoblots of retinal homogenates from m/m and -/- mutant dogs showed no reactivity although the antibody recognizes the mutant protein as demonstrated with heterologously-expressed human B3-D262N (canine numbering). Previous qRT-PCR studies examined expression of both CNGA3 and CNGB3 mRNA. Levels of CNGA3 were similar for unaffected, m/m and -/- retinas; CNGB3 mRNA levels were similar for unaffected and m/m dogs but, as expected, levels in -/- retinas were non-detectable. The affected m/m and -/- dogs provide models for inherited human channelopathies including achromatopsia with the potential for direct retinal examination of affected dogs during development and following gene therapy. Results with m/m dogs show that the CNGB3 subunits are degraded; importantly, both the m/m and -/- dogs loose expression of CNGA3 in outer segments. This surprising result implies that an intact CNGB3 is a requirement for CNGA3 expression, a result not predicted or replicated in heterologous expression of CNGA3 channels.