Abstract
Critical functions of the vascular endothelium are regulated by changes in intracellular [Ca(2+)]. Endothelial dysfunction is tightly associated with cardiovascular disease, and improved understanding of Ca(2+) entry pathways in these cells will have a significant impact on human health. However, much about Ca(2+) influx channels in endothelial cells remains unknown because they are difficult to study using conventional patch-clamp electrophysiology. Here we describe a novel, highly efficient method for recording and analyzing Ca(2+)-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 plasma membrane, most channels are silent even during maximal stimulation with the potent TRPV4 agonist N-((1S)-1-{[4-((2S)-2-{[(2,4-dichlorophenyl)sulfonyl]amino}-3-hydroxypropanoyl)-1-piperazinyl]carbonyl}-3-methylbutyl)-1-benzothiophene-2-carboxamide (GSK1016790A). Furthermore, our findings indicate that GSK1016790A acts by recruiting previously inactive channels, rather than through increasing elevation of basal activity.