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

Bhardwaj, Rajesh; Augustynek, Bartlomiej S.; Ercan-Herbst, Ebru; Kandasamy, Palanivel; Seedorf, Matthias; Peinelt, Christine; Hediger, Matthias A. (2020). Ca2+/Calmodulin Binding to STIM1 Hydrophobic Residues Facilitates Slow Ca2+-Dependent Inactivation of the Orai1 Channel. Cellular physiology and biochemistry : international journal of experimental cellular physiology, biochemistry, and pharmacology, 54(2), pp. 252-270. 10.33594/000000218

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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 (ICRAC). A major regulatory mechanism that prevents deleterious Ca2+ overload is the slow Ca2+-dependent inactivation (SCDI) of ICRAC. 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 ICRAC is as yet unclear.


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.


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 ICRAC, 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 ICRAC caused by Ca2+/CaM, proving a direct role of STIM1 in coordinating SCDI.


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 ICRAC.

Item Type:

Journal Article (Original Article)


04 Faculty of Medicine > Pre-clinic Human Medicine > BioMedical Research (DBMR) > Unit Childrens Hospital > Forschungsgruppe Nephrologie / Hypertonie
04 Faculty of Medicine > Department of Dermatology, Urology, Rheumatology, Nephrology, Osteoporosis (DURN) > Clinic of Nephrology and Hypertension
04 Faculty of Medicine > Pre-clinic Human Medicine > Institute of Biochemistry and Molecular Medicine
04 Faculty of Medicine > Pre-clinic Human Medicine > BioMedical Research (DBMR)

UniBE Contributor:

Bhardwaj, Rajesh; Augustynek, Bartlomiej Stanislaw; Kandasamy, Palanivel; Peinelt, Christine and Hediger, Matthias


600 Technology > 610 Medicine & health
500 Science > 570 Life sciences; biology






Barbara Järmann-Bangerter

Date Deposited:

21 Apr 2020 15:26

Last Modified:

11 Dec 2020 08:42

Publisher DOI:


PubMed ID:


Uncontrolled Keywords:

STIM1; SOCE; ICRAC; SCDI; Calmodulin; Orai1




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