Regulation of Drosophila transient receptor potential-like (TrpL) channels by phospholipase C-dependent mechanisms

• Published in: The Journal of physiology Vol. 530; no. 1; pp. 1 - 19
• Main Authors: Estacion, Mark, Sinkins, William G., Schilling, William P.
• Format: Journal Article
• Language: English
• Published: Oxford, UK The Physiological Society 01.01.2001; Blackwell Science Ltd; Blackwell Science Inc
• Subjects: Animals; Baculoviridae - genetics; Calcium - metabolism; Calmodulin-Binding Proteins - metabolism; Cell Line; CHO Cells; Cricetinae; Drosophila - metabolism; Drosophila Proteins; Fluorescent Dyes; Fura-2; Indicators and Reagents; Membrane Potentials - physiology; Membrane Proteins - metabolism; Oocytes - metabolism; Original; Patch-Clamp Techniques; Phosphatidylinositol 4,5-Diphosphate - metabolism; Spodoptera; Transient Receptor Potential Channels; Type C Phospholipases - metabolism; Xenopus
• ISSN: 0022-3751; 1469-7793
• DOI: 10.1111/j.1469-7793.2001.0001m.x

Patch clamp and fura-2 fluorescence were employed to characterize receptor-mediated activation of recombinant Drosophila TrpL channels expressed in Sf9 insect cells. TrpL was activated by receptor stimulation and by exogenous application of diacylglycerol (DAG) or poly-unsaturated fatty acids (PUFAs). Activation of TrpL was blocked more than 70% by U73122, suggesting that the effect of these agents was dependent upon phospholipase C (PLC). In fura-2 assays, extracellular application of bacterial phosphatidylinositol (PI)-PLC or phosphatidylcholine (PC)-PLC caused a transient increase in TrpL channel activity, the magnitude of which was significantly less than that observed following receptor stimulation. TrpL channels were also activated in excised inside-out patches by cytoplasmic application of mammalian PLC-β2, bacterial PI-PLC and PC-PLC, but not by phospholipase D (PLD). The phospholipases had little or no effect when examined in either whole-cell or cell-attached configurations. TrpL activity was inhibited by addition of phosphatidylinositol-4,5-bisphosphate (PIP 2 ) to excised inside-out membrane patches exhibiting spontaneous channel activity or to patches pre-activated by treatment with PLC. The effect was reversible, specific for PIP 2 , and was not observed with phosphatidylethanolamine (PE), PI, PC or phosphatidylserine (PS). However, antibodies against PIP 2 consistently failed to activate TrpL in inside-out patches. It is concluded that both the hydrolysis of PIP 2 and the generation of DAG are required to rapidly activate TrpL following receptor stimulation, or that some other PLC-dependent mechanism plays a crucial role in the activation process.