Functional Role of Lipid Raft Microdomains in Cyclic Nucleotide-Gated Channel Activation

Abstract

Cyclic nucleotide-gated (CNG) channels are the primary targets of light-and odorant-induced signaling in photoreceptors and olfactory sensory neurons. Compartmentalized cyclic nucleotide signaling is necessary to ensure rapid and efficient activation of these nonselective cation channels. However, relatively little is known about the subcellular localization of CNG channels or the mechanisms of their membrane partitioning. Lipid raft domains are specialized membrane microdomains rich in cholesterol and sphingolipids that have been implicated in the organization of many membrane-associated signaling pathways. Herein, we report that the ␣ subunit of the olfactory CNG channel, CNGA2, associates with lipid rafts in heterologous expression systems and in rat olfactory epithelium. However, CNGA2 does not directly bind caveolin, and its membrane localization overlaps only slightly with that of caveolin at the surface of human embryonic kidney (HEK) 293 cells. To test for a possible functional role of lipid raft association, we treated HEK 293 cells with the cholesterol-depleting agent, methyl-␤-cyclodextrin. Cholesterol depletion abolished prostaglandin E 1 -stimulated CNGA2 channel activity in intact cells. Recordings from membrane patches excised from CNGA2-expressing HEK 293 cells revealed that cholesterol depletion dramatically reduced the apparent affinity of homomeric CNGA2 channels for cAMP but only slightly reduced the maximal current. Our results show that olfactory CNG channels target to lipid rafts and that disruption of lipid raft microdomains dramatically alters the function of CNGA2 channels.

Cytoplasmic cAMP concentrations in intact cardiac myocytes

“…Responses detected by isoforms of these channels that have an EC 50 for activation by cAMP of ϳ10 M suggest that levels can reach high micromolar concentrations (23). Furthermore, because CNG channels appear to be found primarily outside of caveolar membrane fractions (6), this would be consistent with our experimental and modeling results suggesting that cAMP activity is higher specifically in domains associated with extracaveolar and/or bulk cytoplasmic compartments.…”

Pitfalls in isolating lipid rafts

“…1). When investigating the concentration of detergent and incubation time needed to separate raft from non-raft proteins within the range reported in the literature 5,6 , we also observed a dose-response relationship 8 . Future studies should therefore clearly demonstrate that the method selected is appropriate for the tissue type under investigation to separate raft from non-raft proteins when claiming raft association of particular proteins.…”

“…Only a few studies show convincing data of a clear separation of raft from non-raft proteins 5,6,8 . This separation relies highly on the preparation procedure, in particular the use of detergent, for which the type of detergent, the concentration and the duration of incubation are the main determinants.…”

Cell- and subunit-specific mechanisms of CNG channel ciliary trafficking and localization inC. elegans

“…The absence of significant recovery of CNG channel fusion protein fluorescence in AWB and ASK cilia upon photobleaching the middle segments argues against a requirement for IFT in maintaining ciliary localization of these channels in these domains under the examined conditions. One possibility is that these proteins are trafficked through IFT or other mechanisms to cilia middle segments early in development, but are irreversibly anchored once localized, perhaps in association with lipid rafts (Brady et al, 2004;Ding et al, 2008). An alternative hypothesis is that CNG channel turnover in the middle segments is initiated only under defined conditions.…”