Premium
Regulation of Drosophila transient receptor potential‐like (TrpL) channels by phospholipase C‐dependent mechanisms
Author(s) -
Estacion Mark,
Sinkins William G.,
Schilling William P.
Publication year - 2001
Publication title -
the journal of physiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.802
H-Index - 240
eISSN - 1469-7793
pISSN - 0022-3751
DOI - 10.1111/j.1469-7793.2001.0001m.x
Subject(s) - phospholipase c , phosphatidylinositol , transient receptor potential channel , diacylglycerol kinase , microbiology and biotechnology , phospholipase , phosphatidylinositol 4,5 bisphosphate , phosphatidylethanolamine , phosphoinositide phospholipase c , biophysics , stimulation , receptor , biology , chemistry , biochemistry , signal transduction , phosphatidylcholine , protein kinase c , membrane , phospholipid , enzyme , endocrinology
1 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). 2 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. 3 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. 4 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.