Unitary TRPA1‐Mediated Ca2+ Influx Events in Primary Cerebral Artery Endothelial Cells

The sole member of the ankyrin subfamily of transient receptor potential channels, TRPA1, is a Ca 2+ permeable, non‐selective cation channel involved in endothelium‐dependent cerebral artery vasodilation. Total Internal Reflection Fluorescence microscopy (TIRFM) was utilized to investigate the biophysical properties and regulation of TRPA1‐mediated Ca 2+ influx in primary cerebral artery endothelial cells (EC). Rat basilar arteries were cut open and placed intima side down on Matrigel‐coated plates. EC were allowed to proliferate (≤14 days), loaded with Fluo 4‐AM, and imaged in situ using TIRFM. Localized, transient Ca 2+ influx events (mean amplitude (F‐F 0 ) of 0.17±0.02, duration of 0.21±0.03 s, and spatial spread of 2.09±0.29 μm 2 ) were detected at a whole‐cell frequency of 0.04±0.02 Hz (n = 24) under basal conditions. Mean event frequency and duration greatly increased with addition of the TRPA1 agonist allyl isothiocyanate (AITC) (0.23±0.08 Hz, 1.64±0.10 s, n = 25). These increases were attenuated in the presence of the selective TRPA1 antagonist HC‐030031 (0.03±0.02 Hz, 0.65±0.16 s, n = 10 cells). Histogram analysis of AITC‐invoked event amplitudes indicates that these Ca 2+ signals display quantal separation. Similar events were recorded from HEK 293 cells overexpressing TRPA1. Together, these data suggest that quantal, TRPA1‐mediated Ca 2+ influx can be recorded from EC using TIRFM. RO1HL091905.