Native cone photoreceptor cyclic nucleotide‐gated channel is a heterotetrameric complex comprising both CNGA3 and CNGB3: a study using the cone‐dominant retina of Nrl−/− mice

Abstract

Cone vision mediated by photoreceptor cyclic nucleotide-gated (CNG) channel activation is essential for central and color vision and visual acuity. Mutations in genes encoding the cone CNG channel subunits, CNGA3 and CNGB3, have been linked to various forms of achromatopsia and progressive cone dystrophy in humans. This study investigates the biochemical components of native cone CNG channels, using the cone-dominant retina in mice deficient in the transcription factor neural retina leucine zipper (Nrl). Abundant expression of CNGA3 and CNGB3 but no rod CNG channel expression was detected in Nrl-/- retina by western blotting and immunolabeling. Localization of cone CNG channel in both blue (S)- and red/green (M)-cones was shown by double immunolabeling using antibodies against the channel subunits and against the S- and M-opsins. Immunolabeling also showed co-localization of CNGA3 and CNGB3 in the mouse retina. Co-immunoprecipitation demonstrated the direct interaction between CNGA3 and CNGB3. Chemical cross-linking readily generated products at sizes consistent with oligomers of the channel complexes ranging from dimeric to tetrameric complexes, in a concentration- and time-dependent pattern. Thus this work provides the first biochemical evidence showing the inter-subunit interaction between CNGA3 and CNGB3 and the presence of heterotetrameric complexes of the native cone CNG channel in retina. No association between CNGA3 and the cone Na(+)/Ca(2+)-K(+) exchanger (NCKX2) was shown by co-immunoprecipitation and chemical cross-linking. This may implicate a distinct modulatory mechanism for Ca(2+) homeostasis in cones compared to rods.