Background/aims: Store-operated Ca entry (SOCE) through plasma membrane Ca channel Orai1 is essential for many cellular processes. SOCE, activated by ER Ca store-depletion, relies on the gating function of STIM1 Orai1-activating region SOAR of the ER-anchored Ca-sensing protein STIM1. Electrophysiologically, SOCE is characterized as Ca release-activated Ca current (I). A major regulatory mechanism that prevents deleterious Ca overload is the slow Ca-dependent inactivation (SCDI) of I. Several studies have suggested a role of Ca/calmodulin (Ca/CaM) in triggering SCDI. However, a direct contribution of STIM1 in regulating Ca/CaM-mediated SCDI of I is as yet unclear.
Methods: The Ca/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 Ca influx was measured using Fluorometric Imaging Plate Reader and whole-cell patch clamp in cells transfected with STIM1 CaM binding mutants. The involvement of Ca/CaM in SCDI was investigated by including recombinant human CaM in patch pipette in electrophysiology.
Results: Here we identified residues Leu/Val (H1) and Leu/Phe (H2) within SOAR that serve as hydrophobic anchor sites for Ca/CaM binding. The bifunctional H2 site is critical for both Orai1 activation and Ca/CaM binding. Single residue mutations of Phe to less hydrophobic residues significantly diminished SOCE and I, independent of Ca/CaM. Hence, the role of H2 residues in Ca/CaM-mediated SCDI cannot be precisely evaluated. In contrast, the H1 site controls exclusively Ca/CaM binding and subsequently SCDI, but not Orai1 activation. V375A but not V375W substitution eliminated SCDI of I caused by Ca/CaM, proving a direct role of STIM1 in coordinating SCDI.
Conclusion: Taken together, we propose a mechanistic model, wherein binding of Ca/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 Ca/CaM are functionally coordinated to control I.
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