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Review
. 2013 Nov 15;305(10):C999-C1008.
doi: 10.1152/ajpcell.00273.2013. Epub 2013 Sep 11.

TRP channel Ca(2+) sparklets: fundamental signals underlying endothelium-dependent hyperpolarization

Michelle N Sullivan  1 , Scott Earley
Affiliations
Review

TRP channel Ca(2+) sparklets: fundamental signals underlying endothelium-dependent hyperpolarization

Michelle N Sullivan et al. Am J Physiol Cell Physiol. .

Abstract

Important functions of the vascular endothelium, including permeability, production of antithrombotic factors, and control of vascular tone, are regulated by changes in intracellular Ca(2+). The molecular identities and regulation of Ca(2+) influx channels in the endothelium are incompletely understood, in part because of experimental difficulties associated with application of patch-clamp electrophysiology to native endothelial cells. However, advances in confocal and total internal reflection fluorescence microscopy and the development of fast, high-affinity Ca(2+)-binding fluorophores have recently allowed for direct visualization and characterization of single-channel transient receptor potential (TRP) channel Ca(2+) influx events in endothelial cells. These events, called "TRP channel Ca(2+) sparklets," have been optically recorded from primary endothelial cells and the intact endothelium, and the biophysical properties and fundamental significance of these Ca(2+) signals in vasomotor regulation have been characterized. This review will first briefly discuss the role of endothelial cell TRP channel Ca(2+) influx in endothelium-dependent vasodilation, describe improved methods for recording unitary TRP channel activity using optical methods, and highlight discoveries regarding the regulation and physiological significance of TRPV4 Ca(2+) sparklets in the vascular endothelium enabled by this new technology. Perspectives on the potential use of these techniques to evaluate changes in TRP channel Ca(2+) influx activity associated with endothelial dysfunction are offered.

Keywords: Ca2+ sparklet; TRPV4; confocal microscopy; endothelium; total internal reflection fluorescence microscopy.

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Figures

Fig. 1.
Fig. 1.
Visualization of membrane Ca2+ channel activity using total internal reflection fluorescence (TIRF) microscopy. A: when light is directed toward a glass-cell interface at an angle (θ) greater than a critical angle (θc), light becomes totally, internally reflected away from the interface surface. The total internal reflection creates a low-energy plane of illumination (evanescent field) penetrating only ∼100 nm into a cell, which allows for visualization of plasma membrane (PM) Ca2+ channel activity without background cytosolic Ca2+ sources (B). Ca2+ (gray circles) that enters the cell will be bound by fluorescent Ca2+ indicators and produce a fluorescence signal at the site of entry. This technique allows for visualization of Ca2+ channel activity on the entire bottom surface of the cell. ER, endoplasmic reticulum.
Fig. 2.
Fig. 2.
Transient receptor potential vanilloid (TRPV4) Ca2+ sparklets in endothelial cells. A: pseudocolor time lapse of TIRF microscopy images of a primary human microvascular endothelial cell loaded with the Ca2+ indicator fluo 4-AM. Unitary TRPV4 Ca2+ influx events (i.e., TRPV4 Ca2+ sparklets) are observed as transient increases in fluorescence (green). Scale bar, 10 μm. Inset: magnifications of TRPV4 Ca2+ sparklet. Scale bar, 2 μm. B: pseudocolor image of a rat airway smooth muscle cell TRPV4 Ca2+ sparklet (top left; scale bar, 10 μm) and fluorescence signals produced from opening of 1, 2, 3, or 4 TRPV4 channels (bottom left; scale bar, 2 μm). Recording of fluorescence vs. time for region of interest (highlighted in top left) indicates opening of 1–4 TRPV4 channels (right).
Fig. 3.
Fig. 3.
TRPV4 Ca2+ sparklets in endothelial cells (ECs) initiate vasodilation. TRPV4 Ca2+ influx from the extracellular space through as few as 3–4 channels activates Ca2+-activated K+ (KCa) channels, resulting in K+ efflux from the cell. This hyperpolarizes the EC membrane, which directly hyperpolarizes smooth muscle cells (SMCs) via gap junctions located within myoendothelial junctions. IEL, internal elastic lamina; Vm, membrane potential.
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