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Xi-Qin Ding, Alexander Matveev, Anil Singh, Komori Komori, Hiroyuki Matsumoto; Biochemical Characterization of Cone Cyclic Nucleotide-gated Channel. Invest. Ophthalmol. Vis. Sci. 2011;52(14):33.
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Photopic vision is mediated by cone photoreceptor cyclic nucleotide-gated (CNG) channel and is essential for color perception and visual acuity. Cone CNG channel is composed of two structurally related subunit types, CNGA3 and CNGB3. Naturally occurring mutations in the channel subunits are associated with achromatopsia, progressive cone dystrophy, and some maculopathies. Unfortunately, our current understanding of the structure of cone CNG channel is quite limited because of the availability of samples. We have shown that the cone dominant Nrl-/- retina is a valuable model to study cone CNG channel. This work investigates the complex structure and stoichiometry of cone CNG channel using this mouse line.
Cone CNG channel in the mouse retina was analyzed by the infrared fluorescence Western detection. Antibodies against CNGA3 and CNGB3 were labeled with the fluorescence dyes IRDye-800CW and IRDye-680. Infrared fluorescence signals were detected by an Odyssey® Infrared Imaging System. Chemical cross-linking, followed by NuPAGE separation and simultaneous detection of CNGA3 and CNGB3, and blue native-PAGE were performed to determine the components and size of the channel complexes.
CNGA3 and CNGB3 in the mouse retina were detected by the respective antibodies with a linear range (r2 ≥ 0.97). Simultaneous detection of CNGA3 and CNGB3 on the same blots was achieved and the specific detection was shown by peptide competition experiments. The cone CNG channel complexes at varying sizes equivalent to the dimer (~150 kDa), trimer (~240 kDa), and tetramer (~320 kDa) were detected by chemical cross-linking combined with the two-color detections of CNGA3 and CNGB3. The relative signals of CNGB3 were more abundant in the dimeric and trimeric complexes compared to the tetrameric ones. Quantification of the fluorescence intensities of the tetrameric complexes shows that the CNGA3 signal was nearly 3-fold higher than the CNGB3 signal. The channel complexes that were separated on a blue native-PAGE migrated to an area between 300 and 420 kDa, which is close to the results obtained from the chemical cross-linking experiments.
We analyzed the native cone CNG channel complexes using infrared fluorescence detection combined with chemical cross-linking and blue native-PAGE. The experimental results suggest that the cone CNG channel is a heterotetrameric complex likely at a stoichiometry of three CNGA3 and one CNGB3.
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