A calcium/cAMP signaling loop at the ORAI1 mouth drives channel inactivation to shape NFAT induction

James W. Putney,Céline Delierneux,Ryan Yoast,Martin Johnson,Scott Emrich,Trayambak Pathak,Shilan Wu,Nadine Hempel,Donald L. Gill,Maxime Gueguinou,Mohamed Trebak,Robert M. Nwokonko,Ping Xin,Xuexin Zhang

doi:10.1038/s41467-019-09593-0

Abstract

ORAI1 constitutes the store-operated Ca2+ release-activated Ca2+ (CRAC) channel crucial for life. Whereas ORAI1 activation by Ca2+-sensing STIM proteins is known, still obscure is how ORAI1 is turned off through Ca2+-dependent inactivation (CDI), protecting against Ca2+ toxicity. Here we identify a spatially-restricted Ca2+/cAMP signaling crosstalk critical for mediating CDI. Binding of Ca2+-activated adenylyl cyclase 8 (AC8) to the N-terminus of ORAI1 positions AC8 near the mouth of ORAI1 for sensing Ca2+. Ca2+ permeating ORAI1 activates AC8 to generate cAMP and activate PKA. PKA, positioned by AKAP79 near ORAI1, phosphorylates serine-34 in ORAI1 pore extension to induce CDI whereas recruitment of the phosphatase calcineurin antagonizes the effect of PKA. Notably, CDI shapes ORAI1 cytosolic Ca2+ signature to determine the isoform and degree of NFAT activation. Thus, we uncover a mechanism of ORAI1 inactivation, and reveal a hitherto unappreciated role for inactivation in shaping cellular Ca2+ signals and NFAT activation.

Highlights

ORAI1 constitutes the store-operated Ca2+ release-activated Ca2+ (CRAC) channel crucial for life
ORAI1 knockout was confirmed by genomic sequencing, western blot (Supplementary Fig. 1a, b; compare lane 1 with 2 and lane 5 with 6; see Supplementary Fig. 1c), and Fura-2 Ca2+ imaging of Store-operated Ca2+ entry (SOCE) triggered by store depletion using the sarcoplasmic/endoplasmic reticulum Ca2+ ATPase (SERCA) blocker, thapsigargin at 2 μM (Supplementary Fig. 1d, e)
When Ca2+-dependent inactivation (CDI) was measured with 10 mM EGTA in the patch pipette using a voltage-step protocol (Supplementary Fig. 1f; see Methods), CRAC currents mediated by ORAI1 showed robust CDI compared with those mediated by ORAI1β (Fig. 1b, c)

Summary

Introduction

ORAI1 constitutes the store-operated Ca2+ release-activated Ca2+ (CRAC) channel crucial for life. Whereas ORAI1 activation by Ca2+-sensing STIM proteins is known, still obscure is how ORAI1 is turned off through Ca2+-dependent inactivation (CDI), protecting against Ca2+ toxicity. The cellular and physiological outcomes controlled by ORAI1 are determined by net CRAC channel activity, which is shaped by the critical balance between its storedependent activation and its Ca2+-dependent inactivation (CDI). Depletion of Ca2+ from the ER lumen causes the ER Ca2+ sensor protein, stromal interaction molecule 1 (STIM1), to lose Ca2+ binding from a low-affinity luminal EF hand, and gain an extended conformation that interacts with PM lipids within ER-PM junctions where it physically binds and opens ORAI1 to mediate CRAC current[2,9]. CDI is driven by an unknown Ca2+-dependent mechanism located either on the CRAC channel complex or situated a few nanometers from the mouth of the channel, reflecting a role of a highly localized Ca2+ microdomain at the close vicinity of the CRAC channel pore[2]

Methods

Results

Conclusion