The TRPV1 ion channel serves as an integrator of noxious stimuli with its activation linked to pain and neurogenic inflammation. Cholesterol, a major component of cell membranes, modifies the function of several types of ion channels. Here, using measurements of capsaicin-activated currents in excised patches from TRPV1-expressing HEK cells, we show that enrichment with cholesterol, but not its diastereoisomer epicholesterol, markedly decreased wild-type rat TRPV1 currents. Substitutions in the S5 helix, rTRPV1-R579D, and rTRPV1-F582Q, decreased this cholesterol response and rTRPV1-L585I was insensitive to cholesterol addition. Two human TRPV1 variants, with different amino acids at position 585, had different responses to cholesterol with hTRPV1-Ile 585 being insensitive to this molecule. However, hTRPV1-I585L was inhibited by cholesterol addition similar to rTRPV1 with the same S5 sequence. In the absence of capsaicin, cholesterol enrichment also inhibited TRPV1 currents induced by elevated temperature and voltage. These data suggest that there is a cholesterol-binding site in TRPV1 and that the functions of TRPV1 depend on the genetic variant and membrane cholesterol content. The transient receptor potential (TRP)3 family of ion channels is found throughout the animal kingdom and has been shown to subserve numerous functions. One extensively studied member of this family is the TRPV1 (Vanilloid 1) channel. Structurally, TRPV1 is thought to be a tetramer comprised of subunits each with six transmembrane domains (S1-S6), with the putative pore of the channel located between S5 and S6. It also contains large intracellular amino and carboxyl termini that have been shown to be involved both in channel gating and regulation (for review, see Ref.

Aquaporin-4 (AQP4) is the primary water channel in the mammalian brain, particularly abundant in astrocytes, whose plasma membranes normally contain high concentrations of cholesterol. Here we test the hypothesis that the water permeabilities of two naturally occurring isoforms (AQP4-M1 and AQP4-M23) depend on bilayer mechanical/structural properties modulated by cholesterol and phospholipid composition. Osmotic stress measurements were performed with proteoliposomes containing AQP4 and three different lipid mixtures: 1), phosphatidylcholine (PC) and phosphatidylglycerol (PG); 2), PC, PG, with 40 mol % cholesterol; and 3), sphingomyelin (SM), PG, with 40 mol % cholesterol. The unit permeabilities of AQP4-M1 were 3.3 ± 0.4 × 10(-13) cm(3)/s (mean ± SE), 1.2 ± 0.1 × 10(-13) cm(3)/s, and 0.4 ± 0.1 × 10(-13) cm(3)/s in PC:PG, PC:PG:cholesterol, and SM:PG:cholesterol, respectively. The unit permeabilities of AQP4-M23 were 2.1 ± 0.2 × 10(-13) cm(3)/s, 0.8 ± 0.1 × 10(-13) cm(3)/s, and 0.3 ± 0.1 × 10(-13) cm(3)/s in PC:PG, PC:PG:cholesterol, and SM:PG:cholesterol, respectively. Thus, for each isoform the unit permeabilities strongly depended on bilayer composition and systematically decreased with increasing bilayer compressibility modulus and bilayer thickness. These observations suggest that altering lipid environment provides a means of regulating water channel permeability. Such permeability changes could have physiological consequences, because AQP4 water permeability would be reduced by its sequestration into SM:cholesterol-enriched raft microdomains. Conversely, under ischemic conditions astrocyte membrane cholesterol content decreases, which could increase AQP4 permeability.