Abstract
Vascular endothelial transient potential (TRP) channels, located mostly on the plasma membrane of cells, are critical in regulatory and pathophysiological circumstances. The objective of this chapter is to describe several well-established approaches, ranging from function to molecular assays, to investigate the mechanistic role of TRP channels in vascular endothelial cells. We show experimental procedures and representative figures on the following methods: (1) Isolation and culture of vascular endothelial cells, (2) examination of electrophysiological activity of TRP channel by patch-clamping with whole-cell configuration and its function in vascular tone and blood flow by isometric tension and isobaric diameter measurements, and Laser Doppler flowmetry, (3) detection of TRP channel-mediated intracellular Ca2+ imaging by using fluorescent microscopy, and (4) determination of TRP channel interaction by coimmunoprecipitation, double immunofluorescence staining and Förster resonance energy transfer (FRET) detection.
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References
Vay L, Gu C, McNaughton PA (2012) The thermo-TRP ion channel family: properties and therapeutic implications. Br J Pharmacol 165:787–801
Hecquet CM, Zhang M, Mittal M, Vogel SM, Di A et al (2014) Cooperative interaction of trp melastatin channel transient receptor potential (TRPM2) with its splice variant TRPM2 short variant is essential for endothelial cell apoptosis. Circ Res 114:469–479
Brayden JE, Earley S, Nelson MT, Reading S (2008) Transient receptor potential (TRP) channels, vascular tone and autoregulation of cerebral blood flow. Clin Exp Pharmacol Physiol 35:1116–1120
Holzer P, Izzo AA (2014) The pharmacology of TRP channels. Br J Pharmacol 171:2469–2473
Hellmich UA, Gaudet R (2014) Structural biology of TRP channels. Handb Exp Pharmacol 223:963–990
Owsianik G, Talavera K, Voets T, Nilius B (2006) Permeation and selectivity of TRP channels. Annu Rev Physiol 68:685–717
Kwan HY, Huang Y, Yao X (2007) TRP channels in endothelial function and dysfunction. Biochim Biophys Acta 1772:907–914
Mathar I, Vennekens R, Meissner M, Kees F, Van der Mieren G et al (2010) Increased catecholamine secretion contributes to hypertension in TRPM4-deficient mice. J Clin Invest 120:3267–3279
Lo CY, Tjong YW, Ho JC, Siu CW, Cheung SY et al (2014) An upregulation in the expression of vanilloid transient potential channels 2 enhances hypotonicity-induced cytosolic Ca(2)(+) rise in human induced pluripotent stem cell model of Hutchinson-Gillford Progeria. PLoS One 9:e87273
Entin-Meer M, Levy R, Goryainov P, Landa N, Barshack I et al (2014) The transient receptor potential vanilloid 2 cation channel is abundant in macrophages accumulating at the peri-infarct zone and may enhance their migration capacity towards injured cardiomyocytes following myocardial infarction. PLoS One 9:e105055
Hamill OP, Marty A, Neher E, Sakmann B, Sigworth FJ (1981) Improved patch-clamp techniques for high-resolution current recording from cells and cell-free membrane patches. Pflugers Arch 391:85–100
Minamitani H, Okada E (1993) Microscopic laser Doppler velocimeter measuring blood velocity in single microvessel. Keio J Med 42:186–190
Ma X, Qiu S, Luo J, Ma Y, Ngai CY et al (2010) Functional role of vanilloid transient receptor potential 4-canonical transient receptor potential 1 complex in flow-induced Ca2+ influx. Arterioscler Thromb Vasc Biol 30:851–858
Kobori T, Smith GD, Sandford R, Edwardson JM (2009) The transient receptor potential channels TRPP2 and TRPC1 form a heterotetramer with a 2:2 stoichiometry and an alternating subunit arrangement. J Biol Chem 284:35507–35513
Shen B, Cheng KT, Leung YK, Kwok YC, Kwan HY et al (2008) Epinephrine-induced Ca2+ influx in vascular endothelial cells is mediated by CNGA2 channels. J Mol Cell Cardiol 45:437–445
Masters SC (2004) Co-immunoprecipitation from transfected cells. Methods Mol Biol 261:337–350
Herman B, Krishnan RV, Centonze VE (2004) Microscopic analysis of fluorescence resonance energy transfer (FRET). Methods Mol Biol 261:351–370
Latif R, Graves P (2000) Fluorescent probes: looking backward and looking forward. Thyroid 10:407–412
Du J, Ma X, Shen B, Huang Y, Birnbaumer L et al (2014) TRPV4, TRPC1, and TRPP2 assemble to form a flow-sensitive heteromeric channel. FASEB J 28:4677–4685
Ma X, Cao J, Luo J, Nilius B, Huang Y et al (2010) Depletion of intracellular Ca2+ stores stimulates the translocation of vanilloid transient receptor potential 4-c1 heteromeric channels to the plasma membrane. Arterioscler Thromb Vasc Biol 30:2249–2255
Leung PC, Cheng KT, Liu C, Cheung WT, Kwan HY et al (2006) Mechanism of non-capacitative Ca2+ influx in response to bradykinin in vascular endothelial cells. J Vasc Res 43:367–376
Wong CO, Sukumar P, Beech DJ, Yao X (2010) Nitric oxide lacks direct effect on TRPC5 channels but suppresses endogenous TRPC5-containing channels in endothelial cells. Pflugers Arch 460:121–130
Acknowledgments
This work was supported by grants from the Hong Kong Research Grant Committee TBRS T13-706/11, AoE/M-05/12, CUHK478413 and by the China National Science Foundation 31470912.
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Tjong, Y.W., Yao, X. (2018). Methods for Evaluation of Vascular Endothelial Cell Function with Transient Receptor Potential (TRP) Channel Drugs. In: Boheler, K., Gundry, R. (eds) The Surfaceome. Methods in Molecular Biology, vol 1722. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7553-2_13
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DOI: https://doi.org/10.1007/978-1-4939-7553-2_13
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