Local Ca ²⁺ Entry Via Orai1 Regulates Plasma Membrane Recruitment of TRPC1 and Controls Cytosolic Ca ²⁺ Signals Required for Specific Cell Functions

Abstract

Store-operated Ca ²⁺ entry (SOCE) has been associated with two types of channels: CRAC channels that require Orai1 and STIM1 and SOC channels that involve TRPC1, Orai1, and STIM1. While TRPC1 significantly contributes to SOCE and SOC channel activity, abrogation of Orai1 function eliminates SOCE and activation of TRPC1. The critical role of Orai1 in activation of TRPC1-SOC channels following Ca ²⁺ store depletion has not yet been established. Herein we report that TRPC1 and Orai1 are components of distinct channels. We show that TRPC1/Orai1/STIM1-dependent I _SOC, activated in response to Ca ²⁺ store depletion, is composed of TRPC1/STIM1-mediated non-selective cation current and Orai1/STIM1-mediated I _CRAC; the latter is detected when TRPC1 function is suppressed by expression of shTRPC1 or a STIM1 mutant that lacks TRPC1 gating, STIM1( ⁶⁸⁴EE ⁶⁸⁵). In addition to gating TRPC1 and Orai1, STIM1 mediates the recruitment and association of the channels within ER/PM junctional domains, a critical step in TRPC1 activation. Importantly, we show that Ca ²⁺ entry via Orai1 triggers plasma membrane insertion of TRPC1, which is prevented by blocking SOCE with 1 µM Gd ³⁺, removal of extracellular Ca ²⁺, knockdown of Orai1, or expression of dominant negative mutant Orai1 lacking a functional pore, Orai1-E106Q. In cells expressing another pore mutant of Orai1, Orai1-E106D, TRPC1 trafficking is supported in Ca ²⁺-containing, but not Ca ²⁺-free, medium. Consistent with this, I _CRAC is activated in cells pretreated with thapsigargin in Ca ²⁺-free medium while I _SOC is activated in cells pretreated in Ca ²⁺-containing medium. Significantly, TRPC1 function is required for sustained K _Ca activity and contributes to NFκB activation while Orai1 is sufficient for NFAT activation. Together, these findings reveal an as-yet unidentified function for Orai1 that explains the critical requirement of the channel in the activation of TRPC1 following Ca ²⁺ store depletion. We suggest that coordinated regulation of the surface expression of TRPC1 by Orai1 and gating by STIM1 provides a mechanism for rapidly modulating and maintaining SOCE-generated Ca ²⁺ signals. By recruiting ion channels and other signaling pathways, Orai1 and STIM1 concertedly impact a variety of critical cell functions that are initiated by SOCE.

Author Summary

Store-operated Ca ²⁺ entry is present in all cell types and determines sustained cytosolic [Ca ²⁺] increases that are critical for regulating a wide variety of physiological functions. This Ca ²⁺ entry mechanism is activated in response to depletion of Ca ²⁺ in the endoplasmic reticulum (ER). When ER [Ca ²⁺] is decreased, the Ca ²⁺-sensor protein STIM1 aggregates in the ER membrane and moves to regions in the periphery of the cells where it interacts with and activates two major types of channels that contribute to store-operated Ca ²⁺ entry: CRAC and SOC. While gating of Orai1 by STIM1 is sufficient for CRAC channel activity, both Orai1 and transient receptor potential channel 1 (TRPC1) contribute to SOC channel function. The molecular composition of SOC channels and the critical role of Orai1 in activation of TRPC1 have not yet been established. In this study, we demonstrate that TRPC1 and Orai1 are components of distinct channels, both of which are regulated by STIM1. Importantly, we show that Orai1-mediated Ca ²⁺ entry triggers plasma membrane insertion of TRPC1 which is then gated by STIM1. Ca ²⁺ entry via functional TRPC1-STIM1 channels provides additional increase in cytosolic [Ca ²⁺] that is required for regulation of specific cell functions such as K _Ca activation. Together, our findings elucidate the critical role of Orai1 in TRPC1 channel function. We suggest that the regulation of TRPC1 trafficking provides a mechanism for rapidly modulating cytosolic [Ca ²⁺] following Ca ²⁺ store depletion.