Dissociation of regulated trafficking of TRPC3 channels to the plasma membrane from their activation by phospholipase C.
Regulated translocation of canonical transient receptor potential (TRPC) proteins to the plasma membrane has been proposed as a mechanism of their activation. By using total internal reflection fluorescence microscopy (TIRFM), we monitored green fluorescent protein-labeled TRPC3 (TRPC3-GFP) movement to the plasma membrane in HEK293 cells stably expressing this fusion protein. We observed no increase in TRPC3-GFP TIRFM in response to the muscarinic receptor agonist methacholine or the synthetic diacylglycerol, 1-oleoyl-2-acetyl-sn-glycerol, despite activation of TRPC3 by these agents. We did, however, observe a TIRFM response to epidermal growth factor (EGF). This TIRFM response to EGF was accompanied by increased Ba2+ entry and TRPC3 currents. However, 1-oleoyl-2-acetyl-sn-glycerol-induced TRPC3 activity was not increased. TIRFM also increased in response to Gd3+, a competitive inhibitor of TRPC3 channels. This may be indicative of constitutive trafficking of TRPC3, with Gd3+ acting to "trap" cycling TRPC3 molecules in the plasma membrane. Consistent with this interpretation, TRPC3-expressing cells exhibited large variance in membrane capacitance, and this variance was decreased by both Gd3+ and EGF. These results indicate the following: (i) trafficking of TRPC3 may play a role in regulating the concentration of channels in the plasma membrane but is not involved in activation through the phospholipase C pathway; (ii) TRPC3 undergoes constitutive cyclical trafficking in the plasma membrane, and the mechanism by which growth factors increase the number of plasma membrane channels may involve stabilizing them in the plasma membrane.
Smyth
Jeremy T
JT
Laboratory of Signal Transduction, NIEHS, National Institutes of Health, Department of Health and Human Services, Research Triangle Park, North Carolina 27709, USA.
Lemonnier
Loïc
L
Vazquez
Guillermo
G
Bird
Gary S
GS
Putney
James W
JW
D001464:Barium / Q000378:metabolism; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism*; D002478:Cells, Cultured; D004075:Diglycerides / Q000494:pharmacology; D004815:Epidermal Growth Factor / Q000494:pharmacology; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D007668:Kidney / Q000378:metabolism; D016210:Methacholine Chloride / Q000494:pharmacology; D018721:Muscarinic Agonists / Q000494:pharmacology; D021381:Protein Transport; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D050052:TRPC Cation Channels / Q000378:metabolism*; D010738:Type C Phospholipases / Q000378:metabolism*
C011439:1,2-diacylglycerol; D004075:Diglycerides; D018721:Muscarinic Agonists; D011993:Recombinant Fusion Proteins; D050052:TRPC Cation Channels; C104603:TRPC3 cation channel; C485184:enhanced green fluorescent protein; D016210:Methacholine Chloride; D049452:Green Fluorescent Proteins; D001464:Barium; D004815:Epidermal Growth Factor; C038237:1-oleoyl-2-acetylglycerol; D010738:Type C Phospholipases