Dissecting the pathogenic mechanisms of mutations in the pore region of the human cone photoreceptor cyclic nucleotide‐gated channel

The CNGA3 gene encodes the A3 subunit of the cone photoreceptor cyclic nucleotide‐gated (CNG) channel, an essential component of the phototransduction cascade. Certain mutations in CNGA3 cause autosomal recessive achromatopsia, a retinal disorder characterized by severely reduced visual acuity, lack of color discrimination, photophobia, and nystagmus. We identified three novel mutations in the pore‐forming region of CNGA3 (L363P, G367V, and E376K) in patients diagnosed with achromatopsia. We assessed the expression and function of channels with these three new and two previously described mutations (S341P and P372S) in a heterologous HEK293 cell expression system using Western blot, subcellular localization on the basis of immunocytochemistry, calcium imaging, and patch clamp recordings. In this first comparative functional analysis of disease‐associated mutations in the pore of a CNG channel, we found impaired surface expression of S341P, L363P, and P372S mutants and reduced macroscopic currents for channels with the mutations S341P, G367V, and E376K. Calcium imaging and patch clamp experiments after incubation at 37°C revealed nonfunctional homo‐ and heteromeric channels in all five mutants, but incubation at 27°C combined with coexpression of the B3 subunit restored residual function of channels with the mutations S341P, G367V, and E376K. Hum Mutat 31:830–839, 2010. © 2010 Wiley‐Liss, Inc.