STIM1 activates CRAC channels through rotation of the pore helix to open a hydrophobic gate

Abstract

Store-operated Ca2+ release-activated Ca2+ (CRAC) channels constitute a major pathway for Ca2+ influx and mediate many essential signalling functions in animal cells, yet how they open remains elusive. Here, we investigate the gating mechanism of the human CRAC channel Orai1 by its activator, stromal interacting molecule 1 (STIM1). We find that two rings of pore-lining residues, V102 and F99, work together to form a hydrophobic gate. Mutations of these residues to polar amino acids produce channels with leaky gates that conduct ions in the resting state. STIM1-mediated channel activation occurs through rotation of the pore helix, which displaces the F99 residues away from the pore axis to increase pore hydration, allowing ions to flow through the V102-F99 hydrophobic band. Pore helix rotation by STIM1 also explains the dynamic coupling between CRAC channel gating and ion selectivity. This hydrophobic gating mechanism has implications for CRAC channel function, pharmacology and disease-causing mutations.

Pore opening mechanism of CRAC channels

“…3B). Indeed, consistent with its role as a potential gate, substitutions of F99 to more polar residues (Tyr, Ser, Thr, Cys, Met) yielded leaky channels in the absence of STIM1 [51]. Channels with substitutions to Ile, Leu, or Val, however, remained closed.…”

“…Ion sizing experiments in the constitutively conducting V102C mutant showed that STIM1 binding causes the Orai1 channel pore to narrow [39], which suggests that the gating mechanism in CRAC channels does not involve simple pore dilation to permit ion flow. Paradoxically, direct examination of the conformational change during gating using the accessibility of pore-applied thiol reagents revealed that access of the reagents Cd 2+ and Ag + to V102C is not altered by STIM1 binding, indicating that V102C remains pore-facing in both the resting and activated conformations [51]. This surprising finding implies that although V102 likely forms part of the hydrophobic barrier to stabilize closed channels, it alone does not constitute the channel gate given its constant exposure into the pore.…”

“…This difference led us to hypothesize that STIM1 binding evokes a conformational change that alters the accessibility of these residues upon pore opening, thereby exposing G98 towards the pore and concealing F99 away from it. Indeed, analysis of the accessibility of cysteine mutants of key pore residues in the V102A background (to confer constitutive ion conduction in the absence of STIM1) indicated that F99C is blocked strongly by Cd 2+ , but this block decreases dramatically when STIM1 is added [51]. Conversely, the G98C/V102A current is weakly inhibited by Cd 2+ , but the addition of STIM1 greatly increases its sensitivity to Cd 2+ blockade [51].…”

“…Indeed, analysis of the accessibility of cysteine mutants of key pore residues in the V102A background (to confer constitutive ion conduction in the absence of STIM1) indicated that F99C is blocked strongly by Cd 2+ , but this block decreases dramatically when STIM1 is added [51]. Conversely, the G98C/V102A current is weakly inhibited by Cd 2+ , but the addition of STIM1 greatly increases its sensitivity to Cd 2+ blockade [51]. These functional results imply that STIM1 induces a modest (~20°) counterclockwise rotation of the pore helix that moves F99 away from the pore axis but which brings G98 towards a more pore-facing orientation (Fig.…”

“…This observation is consistent with a scenario wherein F99 functions as an energy barrier for water and ion occupancy, with manipulations that lower hydrophobicity at this position evoking leaky channels, whereas maintaining the barrier with hydrophobic amino acid substitutions stabilize the non-conducting state. Surprisingly, the Ile, Leu, and Val mutants remained closed even in the presence of STIM1, but could be opened by decreasing the hydrophobicity at the upstream Val through a V102A substitution [51]. These data indicated that increasing the overall hydrophobicity in this region produces an insurmountable energetic barrier even when STIM1 is bound, thereby stabilizing the closed channel state.…”