Membrane lipid modulations by methyl-ß-cyclodextrin uncouple the Drosophila light-activated phospholipase C from TRP and TRPL channel gating.

ABSTRACT

Sterols are hydrophobic molecules, known to cluster signaling membrane-proteins in lipid rafts, while methyl-ß-cyclodextrin (MßCD) has been a major tool for modulating membrane-sterol content for studying its effect on membrane proteins, including the transient receptor potential (TRP) channels. The Drosophila light-sensitive TRP channels are activated downstream of a G-protein-coupled phospholipase Cß (PLC) cascade. In phototransduction, PLC is an enzyme that hydrolyzes phosphatidylinositol 4,5-bisphosphate (PIP2) generating diacylglycerol, inositol-tris-phosphate, and protons, leading to TRP and TRP-like (TRPL) channel openings. Here, we studied the effects of MßCD on Drosophila phototransduction using electrophysiology while fluorescently monitoring PIP2 hydrolysis, aiming to examine the effects of sterol modulation on PIP2 hydrolysis and the ensuing light-response in the native system. Incubation of photoreceptor cells with MßCD dramatically reduced the amplitude and kinetics of the TRP/TRPL-mediated light response. MßCD also suppressed PLC-dependent TRP/TRPL constitutive channel activity in the dark induced by mitochondrial uncouplers, but PLC-independent activation of the channels by linoleic acid was not affected. Furthermore, MßCD suppressed a constitutively active TRP mutant-channel, trpP365, suggesting that TRP channel activity is a target of MßCD action. Importantly, whole-cell voltage-clamp measurements from photoreceptors and simultaneously monitored PIP2-hydrolysis by translocation of fluorescently tagged Tubby protein domain, from the plasma membrane to the cytosol, revealed that MßCD virtually abolished the light response when having little effect on the light-activated PLC. Together, MßCD uncoupled TRP/TRPL channel gating from light-activated PLC and PIP2-hydrolysis suggesting the involvement of distinct nanoscopic lipid domains such as lipid rafts and PIP2 clusters in TRP/TRPL channel gating.