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

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 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 Cβ (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.

Diacylglycerol Activates the Light Sensitive Channel TRPL Expressed in HEK Cells.

Physiological activation by light of the TRP and TRP-like (TRPL) channels requires the activation of phospholipase Cβ (PLC). The hydrolysis of phosphatidylinositol 4,5, bisphosphate (PIP) by PLC is a crucial step in the still-unclear light activation, while the generation of Diacylglycerol (DAG) by PLC seems to be involved. In this study, we re-examined the ability of a DAG analogue 1-oleoyl-2-acetyl-sn-glycerol (OAG) to activate the TRPL channels expressed in HEK cells.

Electrophysiological Methods for Measuring Photopigment Levels in Drosophila Photoreceptors.

The Drosophila G-protein-coupled photopigment rhodopsin (R) is composed of a protein (opsin) and a chromophore. The activation process of rhodopsin is initiated by photon absorption-inducing isomerization of the chromophore, promoting conformational changes of the opsin and resulting in a second dark-stable photopigment state (metarhodopsin, M). Investigation of this bi-stable photopigment using random mutagenesis requires simple and robust methods for screening mutant flies.

The Role of Membrane Lipids in Light-Activation of TRP Channels.

Transient Receptor Potential (TRP) channels constitute a large superfamily of polymodal channel proteins with diverse roles in many physiological and sensory systems that function both as ionotropic and metabotropic receptors. From the early days of TRP channel discovery, membrane lipids were suggested to play a fundamental role in channel activation and regulation. A prominent example is the TRP and TRP-like (TRPL) channels, which are predominantly expressed in the visual system of .

Modulation of Transient Receptor Potential C Channel Activity by Cholesterol.

Changes of cholesterol level in the plasma membrane of cells have been shown to modulate ion channel function. The proposed mechanisms underlying these modulations include association of cholesterol to a single binding site at a single channel conformation, association to a highly flexible cholesterol binding site adopting multiple poses, and perturbation of lipid rafts. These perturbations have been shown to induce reversible targeting of mammalian transient receptor potential C (TRPC) channels to the cholesterol-rich membrane environment of lipid rafts.

Depletion of Membrane Cholesterol Suppresses Drosophila Transient Receptor Potential-Like (TRPL) Channel Activity.

Cholesterol is an essential compound of higher eukaryotic cell membranes and a known modulator of ion channel activity. Changes in phospholipids and cholesterol composition of cell membranes are known to alter the activity of ion channels. However, there is little knowledge on the effects of cholesterol on transient receptor potential (TRP) channels.

Electrophysiological Method for Whole-cell Voltage Clamp Recordings from Drosophila Photoreceptors.

Whole-cell voltage clamp recordings from Drosophila melanogaster photoreceptors have revolutionized the field of invertebrate visual transduction, enabling the use of D. melanogaster molecular genetics to study inositol-lipid signaling and Transient Receptor Potential (TRP) channels at the single-molecule level. A handful of labs have mastered this powerful technique, which enables the analysis of the physiological responses to light under highly controlled conditions.