title: Signal-dependent hydrolysis of phosphatidylinositol 4,5-bisphosphate without activation of phospholipase C: implications on gating of Drosophila TRPL (transient receptor potential-like) channel.
abstract: In Drosophila, a phospholipase C (PLC)-mediated signaling cascade, couples photo-excitation of rhodopsin to the opening of the transient receptor potential (TRP) and TRP-like (TRPL) channels. A lipid product of PLC, diacylglycerol (DAG), and its metabolites, polyunsaturated fatty acids (PUFAs) may function as second messengers of channel activation. However, how can one separate between the increase in putative second messengers, change in pH, and phosphatidylinositol 4,5-bisphosphate (PI(4,5)P(2)) depletion when exploring the TRPL gating mechanism? To answer this question we co-expressed the TRPL channels together with the muscarinic (M1) receptor, enabling the openings of TRPL channels via G-protein activation of PLC. To dissect PLC activation of TRPL into its molecular components, we used a powerful method that reduced plasma membrane-associated PI(4,5)P(2) in HEK cells within seconds without activating PLC. Upon the addition of a dimerizing drug, PI(4,5)P(2) was selectively hydrolyzed in the cell membrane without producing DAG, inositol trisphosphate, or calcium signals. We show that PI(4,5)P(2) is not an inhibitor of TRPL channel activation. PI(4,5)P(2) hydrolysis combined with either acidification or application of DAG analogs failed to activate the channels, whereas PUFA did activate the channels. Moreover, a reduction in PI(4,5)P(2) levels or inhibition of DAG lipase during PLC activity suppressed the PLC-activated TRPL current. This suggests that PI(4,5)P(2) is a crucial substrate for PLC-mediated activation of the channels, whereas PUFA may function as the channel activator. Together, this study defines a narrow range of possible mechanisms for TRPL gating.
authors: Department of Medical Neurobiology, Institute of Medical Research Israel-Canada, The Edmond and Lily Safra Center for Brain Sciences, Faculty of Medicine of the Hebrew University, Jerusalem 91120, Israel.
authors: Shaya
authors: S
authors: Lev
authors: Ben
authors: B
authors: Katz
authors: Vered
authors: V
authors: Tzarfaty
authors: Baruch
authors: B
authors: Minke
mesh_terms: D000818:Animals; D002462:Cell Membrane; D004075:Diglycerides / Q000235:genetics / Q000378:metabolism; D029721:Drosophila Proteins / Q000235:genetics / Q000378:metabolism*; D004331:Drosophila melanogaster; D004789:Enzyme Activation; D005231:Fatty Acids, Unsaturated / Q000235:genetics / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D015640:Ion Channel Gating / Q000502:physiology*; D019269:Phosphatidylinositol 4,5-Diphosphate / Q000235:genetics / Q000378:metabolism*; D015398:Signal Transduction / Q000502:physiology*; D050051:Transient Receptor Potential Channels / Q000235:genetics / Q000378:metabolism*; D010738:Type C Phospholipases / Q000235:genetics / Q000378:metabolism*
chemical_list: D004075:Diglycerides; D029721:Drosophila Proteins; D005231:Fatty Acids, Unsaturated; D019269:Phosphatidylinositol 4,5-Diphosphate; D050051:Transient Receptor Potential Channels; C074192:trpl protein, Drosophila; D010738:Type C Phospholipases