electrophysiological-method-for-whole-cell-voltage-clamp-recordings

Electrophysiological Method for Whole-cell Voltage Clamp Recordings from <em>Drosophila</em> Photoreceptors

Whole-cell voltage clamp recordings from Drosophila melanogaster photoreceptors have revolutionized the field of invertebrate visual transduction, ...

electrophysiological-methods-for-measuring-photopigment-levels

Electrophysiological Methods for Measuring Photopigment Levels in <em>Drosophila</em> Photoreceptors

The Drosophila G-protein-coupled photopigment rhodopsin (R) is composed of a protein (opsin) and a chromophore. The activation process of rhodopsin is ...

Hebrew University

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. In this study, we explore the effects of cholesterol depletion on the Drosophila photoreceptor channel TRP-like (TRPL), when expressed in tissue culture cells. Depletion of membrane cholesterol with methyl-β-cyclodextrin (MβCD) induced fast (<100s) suppression of spontaneous TRPL channel activity, a typical state of expressed TRPL channels in Drosophila S2 cells. An equally fast suppression of receptor-induced TRPL channel activity in HEK293 cells, downstream of phospholipase C (PLC) activation, was also induced by MβCD. Biochemical experiments showed binding of TRPL to immobilized cholesterol, suggesting direct binding of cholesterol to TRPL. Exploring the effects of several mutations in a putative cholesterol-binding site of TRPL was inconclusive as some did not render the channel insensitive to cholesterol depletion while others rendered the channel inactive. We conclude that (i) cholesterol is essential for TRPL channel activity, (ii) TRPL channels interact with cholesterol, and (iii) the binding site of cholesterol in TRPL differs from the putative binding site of TRPV1. Thus, the fast and strong effects of cholesterol depletion on the TRPL channel activity suggest that cholesterol is an important component of fly photoreceptor signaling membrane.

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

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. 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  TRP and TRP-like (TRPL) channels belong to the TRPC channel subfamily. When the  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.

Modulation of Transient Receptor Potential C Channel Activity by Cholesterol.

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 . 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 (PIP) into Diacylglycerol (DAG) and Inositol 1, 4,5-trisphosphate (IP). 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, PIP depletion or IP-mediated Ca 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  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 PIP 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  TRP channels and present the many advantages of this model system in the exploration of TRP channel activation under physiological conditions.

Rita Gutorov

Department of Medical Neurobiology,
Faculty of Medicine and the Edmond and Lily Safra Center for Brain Sciences (ELSC),
Department of Medical Neurobiology, Faculty of Medicine and the Edmond and Lily Safra Center for Brain Sciences (ELSC)

Hebrew University

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

Electrophysiological Methods for Measuring Photopigment Levels in Drosophila Photoreceptors