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X.-Q. Ding, J. B. Fitzgerald, M. E. McClellan, M. H. Elliott; Functional Role of the Low Buoyant-Density Detergent-Resistant Membranes in Cone Cyclic Nucleotide-Gated Channel Activation. Invest. Ophthalmol. Vis. Sci. 2007;48(13):603.
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© ARVO (1962-2015); The Authors (2016-present)
As a growing number of the channel-linked genetic diseases involve the failure of channels to reach the cell surface and to localize to the plasma membrane properly, the functional role of the detergent-resistant membranes (DRMs) is of considerable importance to understand. This work was designed to investigate the relevance of low buoyant-density DRMs in the cone photoreceptor cyclic nucleotide-gated (CNG) channel (CNGA3) activity.
HEK293 and HeLa cells transfected with mouse CNGA3 cDNA were examined for channel expression and cellular localization by Western blotting and immunocytochemistry. Functional activity of CNGA3 in transfected cells was examined by ratiometric [Ca2+]i measurements using a PTI QuantaMaster spectrofluorometer. The Triton X-100 DRMs were isolated from transfected cells using discontinuous sucrose gradient centrifugation. Co-fractionation of CNGA3 with the DRMs was determined by Western blotting using antibodies against the channel subunit and the DRMs marker caveolin. Functional involvement of the DRMs in channel activation was examined by cholesterol depletion experiments using cholesterol depletion agent methyl-ß-cyclodextrin (MCD). Cholesterol analysis was performed by reverse-phase HPLC of the extracted, nonsaponifiable lipids.
Expression and plasma membrane localization of CNGA3 in the transfected cells were demonstrated biochemically and immunocytochemically. Functional activity of CNGA3 was shown as dose-dependent increase of intracellular calcium concentration in response to 8-pCPT-cGMP stimulation. In sucrose fractionation assays, the channel was co-fractionated with caveolin in the low buoyant-density DRMs fractions although significant amount of CNGA3 was also detected in the high buoyant-densityfractions. MCD treatment led to around 74% depletion of membrane cholesterol and such treatment completely abolished the cGMP-stimulated channel activation.
The cone CNG channel is associated with low buoyant-density DRMs in the heterologous expression system and such association is critical for the channel function. Integrity of the DRMs may play a role in the channel membrane targeting, localization and maintenance of the functional conformation, which are essential for the normal channel function.
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