Molecular Characterization of the Ca2+ Release Activated Ca2+ Current in Native Brain Capillary Endothelial Cells

Abstract

Brain capillary endothelial cells (cECs) form the blood-brain barrier orchestrate on-demand increases in blood flow to serve the brain’s unique metabolic needs. An in vivo imaging study showed that brain capillaries generate an array of dynamic Ca2+ signals primarily dependent on the release of Ca2+ from the endoplasmic reticulum (ER). Little is known about the physiological functions and origins of these signals. Store-operated Ca2+ entry (SOCE) is a fundamental Ca2+ influx mechanism activated by emptying ER Ca2+ stores. Following store depletion, interactions between the ER Ca2+-sensor stromal interaction molecule 1 (STIM1) and Orai1, 2, and/or 3 ion channel subunits on the plasma membrane initiate the Ca2+-release activated Ca2+ current (ICRAC) and SOCE. ICRAC and SOCE in brain cECs are uncharacterized. Therefore, we investigated STIM and Orai expression and the biophysical properties of ICRAC in primary brain cECs. Using droplet digital PCR, we found that native brain cECs express Stim1, Stim2, Orai1, and Orai3 but not Orai2. Conventional whole-cell patch-clamp electrophysiology was used to record ICRAC from freshly isolated brain cECs. Currents were induced by including the slow Ca2+ buffer EGTA in the intracellular solution. In contrast to the strong inward rectification of canonical ICRAC, currents recorded from native brain cECs were dually rectifying. The inward and outward components were blocked by La3+, enhanced with 2-APB, and inhibited with the selective Orai1 channel antagonist Synta66. Both ICRAC components were increased when a divalent-free solution (Ca2+ replaced with Cs+) was used. To gain insight into the molecular composition of CRAC channels in brain cECs, we generated EC-specific knockout (ecKO) Stim1, Orai1, and Orai3 mice. ICRAC was absent in cECs obtained from Stim1-ecKO mice. In contrast, ICRAC recorded from Orai1-ecKO mice displayed strong inward rectification with no outward current and was insensitive to Synta66. ICRAC recorded from Orai3-ecKO mice were also inwardly rectifying but were highly sensitive to Synta66. ICRAC was undetectable in cells from Orai1-Orai3-ecKO mice. These data suggest that native ICRAC in brain cECs requires the expression of Stim1, Orai1, and Orai3. The outward component of native ICRAC in these cells requires the expression of Orai1 and Orai3, but the expression of either Orai1 or Orai3 alone is suffcient to maintain the inward component. R35HL155008, R33NS115132, and P20GM130459 (to SE). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.