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+/Ca2+‐K+ exchanger (NCKX2) was shown by co‐immunoprecipitation and chemical cross‐linking. This may implicate a distinct modulatory mechanism for Ca2+ homeostasis in cones compared to rods.

Light-Driven Calcium Signals in Mouse Cone Photoreceptors

“…8 E-G) (Ding and Weinberg, 2007), it is unclear how this could affect cone [Ca 2ϩ ]. Inconsistent with a major role for the NO-sGC-cGMP pathway in mammalian cones are also the facts that CNG channels could not be detected in cone terminals (Matveev et al, 2008) and that we failed to detect NOS activity in the outer retina.…”

Endoplasmic Reticulum Stress-associated Cone Photoreceptor Degeneration in Cyclic Nucleotide-gated Channel Deficiency

“…Nevertheless, as a unique mammalian cone-dominant model, the Nrl Ϫ/Ϫ mouse line has been widely used for the study of cone biology and disease (26,38,39). We have previously shown that the retinas of Nrl Ϫ/Ϫ mice express an abundant amount of cone CNG channel and lack the expression of the rod channel (25). In this study, we show that the double knock-out mice showed a retinal phenotype similar to that in their respective single knock-out mice, i.e.…”

“…Retinal Protein Sample Preparation, SDS-PAGE, and Western Blot Analysis-Retinal protein sample preparation, SDS-PAGE, and Western blotting were performed as described previously (25). Briefly, retinas were homogenized in homogenization buffer (20 mM HEPES-NaOH, pH 7.4, 1 mM EDTA, 200 mM sucrose, containing protease mixture (Sigma)).…”

Differences in RDS trafficking, assembly and function in cones versus rods: insights from studies of C150S-RDS

“…Mouse mAB against GFAP was purchased from Chemicon International (MAB302, Temecula, CA, USA) and was used at 1 : 1000 for IF. Rabbit polyclonal antibody against the b-subunit of the cone cyclic nucleotide-gated channel (CNGB3) was generously shared by Dr Xi-Qin Ding (University of Oklahoma Health Sciences Center) (46,47) and was used at 1 : 250 for IF. Rabbit polyclonal antibody against cone transducin (GNAT2, 1 : 1000 IF) was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA) (10) as was goat polyclonal antibody against S-opsin (1 : 500 IF).…”