Complex Function of Stim1 in the Activation of Store-Independent Orai Channels

Abstract

Orai proteins contribute to Ca2+ entry pathways through store-dependent, Ca2+ release-activated Ca2+ (CRAC) channels (Orai1), and store-independent, arachidonic acid (AA)-regulated Ca2+ (ARC) or LeukotrieneC4-regulated Ca2+ (LRC) channels (Orai1 and Orai3). Remarkably, although activated by fundamentally different mechanisms, both CRAC and ARC/LRC channels share a requirement for STIM1 expression. To date the role of endoplasmic reticulum-resident STIM1 (ER-STIM1) in the activation of CRAC channels is well appreciated. There is a minor pool of STIM1 at the plasma membrane (PM-STIM1) that was shown to be necessary for ARC current activation in HEK293 cells. Using pharmacological tools targeting AA synthesis and metabolism, Ca2+ imaging, whole-cell and perforated patch clamp electrophysiological recordings we demonstrate that both Orai1 and Orai3 are required for ARC and LRC current activation in both primary vascular smooth muscle cells (VSMCs) and HEK293 cells. Surprisingly, while PM-STIM1 is required for ARC and LRC current activation under whole cell patch clamp recordings in both cell types, ER-STIM1 is sufficient for both ARC and LRC channel activation when intact cells are considered. These results are first to demonstrate ARC channel function in primary VSMCs, highlight the complexity of STIM1 regulation of store-independent Orai channels and demonstrate that ARC and LRC currents are mediated by the same channels.