Regulation of olfactory chloride and cyclic nucleotide-gated channels by cholesterol

Odorants in the inhaled air bind to odorant receptors embedded in cilia of olfactory receptor neurons, triggering the opening of the two ciliary olfactory transduction channels; first, a cationic heterotetrameric cAMP-gated channel, followed by an excitatory Ca2+-activated Cl- channel TMEM16B (also called anoctamin 2), which gives rise to a transduction current. While cholesterol is known to modulate ion channels, its effect on the olfactory channels, specifically TMEM16B, is unclear. We addressed how membrane cholesterol regulates these transduction channels. We heterologously expressed the main subunit of the olfactory cyclic nucleotide-gated (CNG) channel, CNGA2, and the Cl- channel TMEM16B and recorded their function in control, cholesterol-depleted, and cholesterol-enriched membranes. Maximal cAMP- or Ca2+-evoked currents were not altered by varying cholesterol levels. TMEM16B showed a progressive reduction in current upon repeated exposure to Ca2+ ("rundown"), which was accelerated in cholesterol-depleted membranes but decelerated in cholesterol-enriched patches when compared with control patches. Maximal cAMP-gated currents were stable over time, but their sensitivity was altered, with lower and higher cholesterol levels increasing and decreasing the channel's sensitivity, respectively. Cholesterol depletion of patches excised from mouse olfactory cilia containing native channels entirely abolished the TMEM16B current, while the maximal current of the native CNG channel remained unaltered. Reduced cholesterol did not change the sensitivity of the native CNG channel. Thus, while both olfactory transduction channels are sensitive to changes in membrane cholesterol in differing ways, the precise channel stoichiometry and the specific membrane environment also contribute to their function.