Functional Analysis of Mutant CNGA3 Channels Associated With Achromatopsia

Abstract

Abstract: : Purpose: Achromatopsia is a rare autosomal recessive inherited disorder which is characterized by reduced visual acuity, lack of color discrimination, photophobia and nystagmus. Mutations in CNGA3, a gene that codes for the cone cyclic nucleotide–gated channel A–subunit, are associated with complete and incomplete forms of achromatopsia. The aim of this study was to investigate the functional activity of mutant CNGA3 channels in a heterologous expression system. Methods: Mutant CNGA3 expression constructs based on the pcDNA3.1 vector were generated by means of in vitro mutagenesis using a wildtype full–length cDNA construct as template. Purified plasmid DNA was transiently expressed in HEK293 cells and the function of the resulting channels analysed by calcium imaging using the calcium–sensitive fluorescent dye Fura–2. Fluorescence was recorded prior to and after induction with the cGMP–analog 8–Br–cGMP. Results: Out of 13 mutant CNGA3 channels, only those with the mutations R427C, R563H and R563C proved to be functional when analysed with calcium imaging. While R427C is located in the linker region between transmembrane helix 6 and the cGMP–binding domain, the other two mutations (R563H and R563C) are situated within the cGMP–binding domain in the linker between strands ß6 and ß7. Conclusions: The CNGA3 mutations R427C, R563H and R563C have been found to occur only in patients with incomplete achromatopsia or patients who have not been classified yet. This is consistent with the observed functionality of heterolog expressed channels with these mutations. Calcium imaging analysis of mutant CNGA3 channels proved to be a valuable method to assess their functional activity and to differentiate between alleles associated with incomplete and complete achromatopsia.

Keywords: ion channels • mutations • photoreceptors