Functional analysis of rod monochromacy-associated missense mutations in the CNGA3 subunit of the cone photoreceptor cGMP-gated channel

Thirty-nine missense mutations, which had been identified in rod monochromacy or related disorders, in the CNGA3 subunit of cone photoreceptor cGMP-gated channels were analyzed. HEK293 cells were transfected with cDNA of the human CNGA3 subunit harboring each of these mutations in an expression vector. Patch–clamp recordings demonstrated that 32 of the 39 mutants did not show cGMP-activated current, suggesting that these 32 mutations cause a loss of function of the channels. From the remaining 7 mutants that showed cGMP-activated current, two mutations in the cyclic nucleotide-binding domain, T565M or E593K, were further studied. The half-maximal activating concentration (K1/2) for cGMP in the homomeric CNGA3-T565M channels (160 μM) was 17.8-fold higher than that of the homomeric wild-type CNGA3 channels (9.0 μM). Conversely, the K1/2 for cGMP in the homomeric CNGA3-E593K channels (3.0 μM) was 3-fold lower than that of the homomeric wild-type CNGA3 channels. These results suggest that the T565M and E593K mutations alter the apparent affinity for cGMP of the channels to cause cone dysfunction, resulting in rod monochromacy.