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PMID: 16129772 Published · ppublish English Journal Article Research Support, N.I.H., Extramural Research Support, U.S. Gov't, P.H.S.

Phospholipase C in living cells: activation, inhibition, Ca2+ requirement, and regulation of M current.

The Journal of general physiology ·Vol. 126 ·No. 3 ·2005-09-00 ·Pages 243-62

Horowitz LF, Hirdes W, Suh BC, Hilgemann DW, Mackie K, Hille B

Abstract

We have further tested the hypothesis that receptor-mediated modulation of KCNQ channels involves depletion of phosphatidylinositol 4,5-bisphosphate (PIP2) by phosphoinositide-specific phospholipase C (PLC). We used four parallel assays to characterize the agonist-induced PLC response of cells (tsA or CHO cells) expressing M1 muscarinic receptors: translocation of two fluorescent probes for membrane lipids, release of calcium from intracellular stores, and chemical measurement of acidic lipids. Occupation of M1 receptors activates PLC and consumes cellular PIP2 in less than a minute and also partially depletes mono- and unphosphorylated phosphoinositides. KCNQ current is simultaneously suppressed. Two inhibitors of PLC, U73122 and edelfosine (ET-18-OCH3), can block the muscarinic actions completely, including suppression of KCNQ current. However, U73122 also had many side effects that were attributable to alkylation of various proteins. These were mimicked or occluded by prior reaction with the alkylating agent N-ethylmaleimide and included block of pertussis toxin-sensitive G proteins and effects that resembled a weak activation of PLC or an inhibition of lipid kinases. By our functional criteria, the putative PLC activator m-3M3FBS did stimulate PLC, but with a delay and an irregular time course. It also suppressed KCNQ current. The M1 receptor-mediated activation of PLC and suppression of KCNQ current were stopped by lowering intracellular calcium well below resting levels and were slowed by not allowing intracellular calcium to rise in response to PLC activation. Thus calcium release induced by PLC activation feeds back immediately on PLC, accelerating it during muscarinic stimulation in strong positive feedback. These experiments clarify important properties of receptor-coupled PLC responses and their inhibition in the context of the living cell. In each test, the suppression of KCNQ current closely paralleled the expected fall of PIP2. The results are described by a kinetic model.

MeSH Terms
Alkylation Animals CHO Cells Calcium/pharmacology Cricetinae Enzyme Activation/drug effects Estrenes/pharmacology Ethylmaleimide/pharmacology Humans Inositol Phosphates/metabolism KCNQ Potassium Channels KCNQ1 Potassium Channel Membrane Potentials/drug effects Muscarinic Agonists/pharmacology Oxotremorine/analogs & derivatives,pharmacology Phosphatidylinositol 4,5-Diphosphate/metabolism Phosphatidylinositol Diacylglycerol-Lyase/antagonists & inhibitors,metabolism Phospholipid Ethers/pharmacology Potassium Channels, Voltage-Gated/antagonists & inhibitors,drug effects,metabolism Protein Kinase C/genetics Pyrrolidinones/pharmacology Receptor, Muscarinic M1/drug effects,genetics,metabolism Sulfonamides/pharmacology Time Factors Transfection
Chemicals
2,4,6-trimethyl-N-(meta-3-trifluoromethylphenyl)benzenesulfonamide Estrenes Inositol Phosphates KCNQ Potassium Channels KCNQ1 Potassium Channel KCNQ1 protein, human Muscarinic Agonists Phosphatidylinositol 4,5-Diphosphate Phospholipid Ethers Potassium Channels, Voltage-Gated Pyrrolidinones Receptor, Muscarinic M1 Sulfonamides 1-(6-((3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl)-1H-pyrrole-2,5-dione edelfosine inositol 1,2,3-trisphosphate Oxotremorine oxotremorine M Protein Kinase C Phosphatidylinositol Diacylglycerol-Lyase Ethylmaleimide Calcium
Authors & Affiliations
6 authors, click to expand affiliations / ORCID
Horowitz Lisa F
Department of Physiology and Biophysics, University of Washington School of Medicine, Seattle 98195, USA.
Hirdes Wiebke
Suh Byung-Chang
Hilgemann Donald W
Mackie Ken
Hille Bertil
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Article Info
Journal
The Journal of general physiology
Abbr.
J Gen Physiol
ISSN
0022-1295
DOI
10.1085/jgp.200509309
Published
2005-09-00
Pages
243-62
Language
English
Region
United States
NLM ID
2985110R
PMCID
PMC2266577
Subset
IM
Grants
NINDS NIH HHS · R01 NS008174 · United States
NHLBI NIH HHS · HL067942 · United States
NIDA NIH HHS · K02 DA000286 · United States
NINDS NIH HHS · NS08174 · United States
NIDA NIH HHS · DA00286 · United States
NINDS NIH HHS · R37 NS008174 · United States
NHLBI NIH HHS · R01 HL067942 · United States
NIAMS NIH HHS · R01 AR017803 · United States
NIDA NIH HHS · R01 DA011322 · United States
NIDA NIH HHS · DA11322 · United States
NIAMS NIH HHS · AR17803 · United States
NINDS NIH HHS · T32 NS007332 · United States
NINDS NIH HHS · NS07332 · United States
External Links
PubMed source DOI: 10.1085/jgp.200509309 PMC Full Text View journal details
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