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 cholesterolrich membrane environment of lipid rafts. Thus, the observed inhibition of TRPC channels by methyl-β-cyclodextrin (MβCD), which induces cholesterol efflux from the plasma membrane, may result from disruption of lipid rafts. This perturbation was also shown to disrupt multimolecular signaling complexes containing TRPC channels. The Drosophila TRP and TRP-like (TRPL) channels belong to the TRPC channel subfamily. When the Drosophila TRPL channel was expressed in S2 or HEK293 cells and perfused with MβCD, the TRPL current was abolished in less than 100 s, fitting well the fast kinetic phase of cholesterol sequestration experiments in cells. It was thus suggested that the fast kinetics of TRPL channel suppression by MβCD arise from disruption of lipid rafts. Accordingly, lipid raft perturbation by cholesterol sequestration could give clues to the function of lipid environment in TRPC channel activity and its mechanism.
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. This technique allows control over the intracellular and extracellular media; the membrane voltage; and the fast application of pharmacological compounds, such as a variety of ionic or pH indicators, to the intra- and extracellular media. With an exceptionally high signal-to-noise ratio, this method enables the measurement of dark spontaneous and light-induced unitary currents (i.e. spontaneous and quantum bumps) and macroscopic Light-induced Currents (LIC) from single D. melanogaster photoreceptors. This protocol outlines, in great detail, all the key steps necessary to perform this technique, which includes both electrophysiological and optical recordings. The fly retina dissection procedure for the attainment of intact and viable ex vivo isolated ommatidia in the bath chamber is described. The equipment needed to perform whole-cell and fluorescence imaging measurements are also detailed. Finally, the pitfalls in using this delicate preparation during extended experiments are explained.
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 Drosophila TRP and TRP-like (TRPL) channels, which are predominantly expressed in the visual system of Drosophila. Light activation of the TRP and TRPL channels, the founding members of the TRP channel superfamily, requires activation of phospholipase Cβ (PLC), which hydrolyzes phosphatidylinositol 4,5-bisphosphate (PIP2) into Diacylglycerol (DAG) and Inositol 1, 4,5-trisphosphate (IP3). However, the events required for channel gating downstream of PLC activation are still under debate and led to several hypotheses regarding the mechanisms by which lipids gate the channels. Despite many efforts, compelling evidence of the involvement of DAG accumulation, PIP2 depletion or IP3-mediated Ca2+ release in light activation of the TRP/TRPL channels are still lacking. Exogeneous application of poly unsaturated fatty acids (PUFAs), a product of DAG hydrolysis was demonstrated as an efficient way to activate the Drosophila TRP/TRPL channels. However, compelling evidence for the involvement of PUFAs in physiological light-activation of the TRP/TRPL channels is still lacking. Light-induced mechanical force generation was measured in photoreceptor cells prior to channel opening. This mechanical force depends on PLC activity, suggesting that the enzymatic activity of PLC converting PIP2 into DAG generates membrane tension, leading to mechanical gating of the channels. In this review, we will present the roles of membrane lipids in light activation of Drosophila TRP channels and present the many advantages of this model system in the exploration of TRP channel activation under physiological conditions.
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