Functional role of lipid raft microdomains in cyclic nucleotide-gated channel activation

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 alpha 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-beta-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.