Vasoconstriction resulting from dynamic membrane trafficking of TRPM4 in vascular smooth muscle cells.
The melastatin (M) transient receptor potential (TRP) channel TRPM4 mediates pressure and protein kinase C (PKC)-induced smooth muscle cell depolarization and vasoconstriction of cerebral arteries. We hypothesized that PKC causes vasoconstriction by stimulating translocation of TRPM4 to the plasma membrane. Live-cell confocal imaging and fluorescence recovery after photobleaching (FRAP) analysis was performed using a green fluorescent protein (GFP)-tagged TRPM4 (TRPM4-GFP) construct expressed in A7r5 cells. The surface channel was mobile, demonstrating a FRAP time constant of 168 +/- 19 s. In addition, mobile intracellular trafficking vesicles were readily detected. Using a cell surface biotinylation assay, we showed that PKC activation with phorbol 12-myristate 13-acetate (PMA) increased (approximately 3-fold) cell surface levels of TRPM4-GFP protein in <10 min. Similarly, total internal reflection fluorescence microscopy demonstrated that stimulation of PKC activity increased (approximately 3-fold) the surface fluorescence of TRPM4-GFP in A7r5 cells and primary cerebral artery smooth muscle cells. PMA also caused an elevation of cell surface TRPM4 protein levels in intact arteries. PMA-induced translocation of TRPM4 to the plasma membrane was independent of PKCalpha and PKCbeta activity but was inhibited by blockade of PKCdelta with rottlerin. Pressure-myograph studies of intact, small interfering RNA (siRNA)-treated cerebral arteries demonstrate that PKC-induced constriction of cerebral arteries requires expression of both TRPM4 and PKCdelta. In addition, pressure-induced arterial myocyte depolarization and vasoconstriction was attenuated in arteries treated with siRNA against PKCdelta. We conclude that PKCdelta activity causes smooth muscle depolarization and vasoconstriction by increasing the number of TRPM4 channels in the sarcolemma.
Crnich
Rachael
R
Vascular Physiology Research Group, Department of Biomedical Sciences, Colorado State University, Fort Collins, Colorado, USA.
Amberg
Gregory C
GC
Leo
M Dennis
MD
Gonzales
Albert L
AL
Tamkun
Michael M
MM
Jaggar
Jonathan H
JH
Earley
Scott
S
D000818:Animals; D002462:Cell Membrane / Q000378:metabolism; D002478:Cells, Cultured; D002536:Cerebral Arteries / Q000166:cytology / Q000378:metabolism; D020536:Enzyme Activators / Q000494:pharmacology; D008297:Male; D008564:Membrane Potentials; D051379:Mice; D009119:Muscle Contraction; D009131:Muscle, Smooth, Vascular / Q000378:metabolism*; D032389:Myocytes, Smooth Muscle / Q000378:metabolism*; D051745:Protein Kinase C-delta / Q000502:physiology; D021381:Protein Transport; D051381:Rats; D017207:Rats, Sprague-Dawley; D050053:TRPM Cation Channels / Q000378:metabolism*; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
D020536:Enzyme Activators; D050053:TRPM Cation Channels; C476766:TRPM4 protein, mouse; D051745:Protein Kinase C-delta; D013755:Tetradecanoylphorbol Acetate