Regulation of Drosophila transient receptor potential-like (TrpL) channels by phospholipase C-dependent mechanisms.
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-b2, 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 (PIP2) 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 PIP2, and was not observed with phosphatidylethanolamine (PE), PI, PC or phosphatidylserine (PS). However, antibodies against PIP2 consistently failed to activate TrpL in inside-out patches. It is concluded that both the hydrolysis of PIP2 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.
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Rammelkamp Center for Education and Research, MetroHealth Medical Center and the Department of Physiology and Biophysics, Case Western Reserve University School of Medicine, Cleveland, OH 44109, USA.
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Schilling
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D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D016466:CHO Cells; D002118:Calcium / Q000378:metabolism; D002148:Calmodulin-Binding Proteins / Q000378:metabolism*; D002460:Cell Line; D006224:Cricetinae; D004330:Drosophila / Q000378:metabolism*; D029721:Drosophila Proteins; D005456:Fluorescent Dyes; D016257:Fura-2; D007202:Indicators and Reagents; D008564:Membrane Potentials / Q000502:physiology; D008565:Membrane Proteins / Q000378:metabolism*; D009865:Oocytes / Q000378:metabolism; D018408:Patch-Clamp Techniques; D019269:Phosphatidylinositol 4,5-Diphosphate / Q000378:metabolism; D018411:Spodoptera; D050051:Transient Receptor Potential Channels; D010738:Type C Phospholipases / Q000378:metabolism*; D014981:Xenopus
D002148:Calmodulin-Binding Proteins; D029721:Drosophila Proteins; D005456:Fluorescent Dyes; D007202:Indicators and Reagents; D008565:Membrane Proteins; D019269:Phosphatidylinositol 4,5-Diphosphate; D050051:Transient Receptor Potential Channels; C074192:trpl protein, Drosophila; D010738:Type C Phospholipases; D002118:Calcium; D016257:Fura-2