A series of Orai1 gating checkpoints in transmembrane and cytosolic regions requires clearance for CRAC channel opening

Abstract

The initial activation step in gating of ubiquitously expressed Orai1 Calcium (Ca2+) ion channels represents the store-dependent coupling to the Ca2+ sensor protein STIM1. An array of constitutively active Orai1 mutants gave rise to the hypothesis that STIM1 mediated Orai1 pore opening is accompanied by a global conformational change of all Orai TM helices within the channel complex. Here, we prove that a local conformational change spreads omnidirectionally within the Orai1 complex. Our results demonstrate that a global, opening-permissive allosteric communication of TM helices is indispensable for pore opening and requires clearance of a series of Orai1 gating checkpoints. We discovered these gating checkpoints in middle and cytosolic extended TM domain regions. Our findings are based on a library of double point mutants that contain each one loss-of-function (LoF) with one gain-of-function (GoF) point mutation in a series of possible combinations. We demonstrated that an array of LoF mutations act dominant over most GoF mutations within the same as well as of an adjacent Orai subunit. We further established inter- and intramolecular salt-bridge interactions of Orai subunits as a core element of an opening-permissive Orai channel architecture. Collectively, clearance and synergistic action of all these gating checkpoints is required to allow STIM1 coupling and Orai1 pore opening. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=166 SRC="FIGDIR/small/207183v2_ufig1.gif" ALT="Figure 1"> View larger version (76K): org.highwire.dtl.DTLVardef@3f80f1org.highwire.dtl.DTLVardef@12a6a0aorg.highwire.dtl.DTLVardef@197b825org.highwire.dtl.DTLVardef@1bab109_HPS_FORMAT_FIGEXP M_FIG C_FIG