Ca2+/Calmodulin Binding to STIM1 Hydrophobic Residues Facilitates Slow Ca2+-Dependent Inactivation of the Orai1 Channel

Abstract

Background/aims: Store-operated Ca²⁺ entry (SOCE) through plasma membrane Ca²⁺ channel Orai1 is essential for many cellular processes. SOCE, activated by ER Ca²⁺ store-depletion, relies on the gating function of STIM1 Orai1-activating region SOAR of the ER-anchored Ca²⁺-sensing protein STIM1. Electrophysiologically, SOCE is characterized as Ca²⁺ release-activated Ca²⁺ current (I_CRAC). A major regulatory mechanism that prevents deleterious Ca²⁺ overload is the slow Ca²⁺-dependent inactivation (SCDI) of I_CRAC. Several studies have suggested a role of Ca²⁺/calmodulin (Ca²⁺/CaM) in triggering SCDI. However, a direct contribution of STIM1 in regulating Ca²⁺/CaM-mediated SCDI of I_CRAC is as yet unclear.

Methods: The Ca²⁺/CaM binding to STIM1 was tested by pulling down recombinant GFP-tagged human STIM1 C-terminal fragments on CaM sepharose beads. STIM1 was knocked out by CRISPR/Cas9 technique in HEK293 cells stably overexpressing human Orai1. Store-operated Ca²⁺ influx was measured using Fluorometric Imaging Plate Reader and whole-cell patch clamp in cells transfected with STIM1 CaM binding mutants. The involvement of Ca²⁺/CaM in SCDI was investigated by including recombinant human CaM in patch pipette in electrophysiology.

Results: Here we identified residues Leu³⁷⁴/Val³⁷⁵ (H1) and Leu³⁹⁰/Phe³⁹¹ (H2) within SOAR that serve as hydrophobic anchor sites for Ca²⁺/CaM binding. The bifunctional H2 site is critical for both Orai1 activation and Ca²⁺/CaM binding. Single residue mutations of Phe³⁹¹ to less hydrophobic residues significantly diminished SOCE and I_CRAC, independent of Ca²⁺/CaM. Hence, the role of H2 residues in Ca²⁺/CaM-mediated SCDI cannot be precisely evaluated. In contrast, the H1 site controls exclusively Ca²⁺/CaM binding and subsequently SCDI, but not Orai1 activation. V375A but not V375W substitution eliminated SCDI of I_CRAC caused by Ca²⁺/CaM, proving a direct role of STIM1 in coordinating SCDI.

Conclusion: Taken together, we propose a mechanistic model, wherein binding of Ca²⁺/CaM to STIM1 hydrophobic anchor residues, H1 and H2, triggers SCDI by disrupting the functional interaction between STIM1 and Orai1. Our findings reveal how STIM1, Orai1, and Ca²⁺/CaM are functionally coordinated to control I_CRAC.

Keywords: STIM1; SOCE; ICRAC; SCDI; Calmodulin; Orai1.