RT Journal Article
SR Electronic
T1 Optical Recording Reveals Novel Properties of GSK1016790A-Induced TRPV4 Channel Activity in Primary Human Endothelial Cells
JF Molecular Pharmacology
JO Mol Pharmacol
FD American Society for Pharmacology and Experimental Therapeutics
SP mol.112.078584
DO 10.1124/mol.112.078584
A1 Michelle N. Sullivan
A1 Michael Francis
A1 Natalie L. Pitts
A1 Mark S. Taylor
A1 Scott Earley
YR 2012
UL http://molpharm.aspetjournals.org/content/early/2012/06/11/mol.112.078584.abstract
AB Critical functions of the vascular endothelium are regulated by changes in intracellular [Ca2+]. Endothelial dysfunction is tightly associated with cardiovascular disease, and improved understanding of Ca2+ entry pathways in these cells will have a significant impact on human health. However, much remains unknown about Ca2+ influx channels in endothelial cells because they are difficult to study using conventional patch clamp electrophysiology. Here we describe a novel, highly efficient method for recording and analyzing Ca2+-permeable channel activity in primary human endothelial cells using a unique combination of Total Internal Reflection Fluorescence Microscopy (TIRFM), custom software-based detection, and selective pharmacology. Our findings indicate that activity of the vanilloid (V) transient receptor potential (TRP) channel TRPV4 can be rapidly recorded and characterized at the single-channel level using this method, providing novel insight into channel function. Using this method, we show that although TRPV4 protein is evenly distributed throughout the cell, most channels are silent even during maximal stimulation with the potent TRPV4 agonist GSK1016790A. Furthermore, our findings indicate that GSK1016790A acts by recruiting previously inactive channels, rather than through increasing basal activity.