Membrane lipid modulations by methyl-β-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-β-cyclodextrin (MβCD) has been a major tool for modulating membrane-sterol content to study 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 Cβ (PLC) cascade. In phototransduction, PLC is a critical enzyme that hydrolyzes phosphatidylinositol 4,5-bisphosphate (PIP2) generating diacylglycerol (DAG), inositol-tris-phosphate (IP3) and protons, leading to TRP and TRP-like (TRPL) channel openings. Here we studied the effects of MβCD 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 MβCD dramatically reduced the amplitude and kinetics of the TRP/TRPL-mediated light-response. MβCD 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, MβCD suppressed a constitutively-active TRP mutant-channel, trpP365, suggesting that TRP channel activity is a target of MβCD action. Importantly, whole-cell voltage-clamp measurements from photoreceptor cells and simultaneously monitored PIP2 hydrolysis by translocation of fluorescently-tagged lipid-binding-Tubby-protein domain, from the plasma membrane to the cytosol revealed that MβCD virtually abolished the light-response when having only little effect on the light-activated PIP2 hydrolysis by PLC. Together, MβCD uncoupled TRP/TRPL channel gating from light-activated PLC and PIP2-hydrolysis suggest the involvement of distinct nanoscopic lipid domains such as lipid-rafts and PIP2-clusters in TRP/TRPL channel gating.