Vasoconstriction resulting from dynamic membrane trafficking of TRPM4 in vascular smooth muscle cells

Rachael Crnich, Gregory C. Amberg, Marie Dennis Leo, Albert L. Gonzales, Michael M. Tamkun, Jonathan Jaggar, Scott Earley

Research output: Contribution to journalArticle

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Abstract

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 (∼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 (∼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 PKCα and PKCβ activity but was inhibited by blockade of PKCδ 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 PKCδ. In addition, pressure-induced arterial myocyte depolarization and vasoconstriction was attenuated in arteries treated with siRNA against PKCδ. We conclude that PKCδ activity causes smooth muscle depolarization and vasoconstriction by increasing the number of TRPM4 channels in the sarcolemma.

Original languageEnglish (US)
JournalAmerican Journal of Physiology - Cell Physiology
Volume299
Issue number3
DOIs
StatePublished - Sep 1 2010

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Vasoconstriction
Vascular Smooth Muscle
Protein Kinase C
Smooth Muscle Myocytes
Membranes
Cerebral Arteries
Green Fluorescent Proteins
Fluorescence Recovery After Photobleaching
Acetates
Small Interfering RNA
Arteries
Cell Membrane
Biotinylation
Transient Receptor Potential Channels
Pressure
Sarcolemma
Fluorescence Microscopy
Constriction
Muscle Cells
Smooth Muscle

All Science Journal Classification (ASJC) codes

  • Cell Biology
  • Physiology
  • Medicine(all)

Cite this

Vasoconstriction resulting from dynamic membrane trafficking of TRPM4 in vascular smooth muscle cells. / Crnich, Rachael; Amberg, Gregory C.; Leo, Marie Dennis; Gonzales, Albert L.; Tamkun, Michael M.; Jaggar, Jonathan; Earley, Scott.

In: American Journal of Physiology - Cell Physiology, Vol. 299, No. 3, 01.09.2010.

Research output: Contribution to journalArticle

Crnich, Rachael ; Amberg, Gregory C. ; Leo, Marie Dennis ; Gonzales, Albert L. ; Tamkun, Michael M. ; Jaggar, Jonathan ; Earley, Scott. / Vasoconstriction resulting from dynamic membrane trafficking of TRPM4 in vascular smooth muscle cells. In: American Journal of Physiology - Cell Physiology. 2010 ; Vol. 299, No. 3.
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AB - 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 (∼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 (∼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 PKCα and PKCβ activity but was inhibited by blockade of PKCδ 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 PKCδ. In addition, pressure-induced arterial myocyte depolarization and vasoconstriction was attenuated in arteries treated with siRNA against PKCδ. We conclude that PKCδ activity causes smooth muscle depolarization and vasoconstriction by increasing the number of TRPM4 channels in the sarcolemma.

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