Ca 2+/Calmodulin Binding to STIM1 Hydrophobic Residues Facilitates Slow Ca 2+-Dependent Inactivation of the Orai1 Channel

Cell Physiol Biochem. 2020 Mar 17;54(2):252-270. doi: 10.33594/000000218.

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 (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.

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 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.

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 ICRAC.

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

MeSH terms

  • CRISPR-Cas Systems
  • Calcium / metabolism*
  • Calcium Channels / genetics
  • Calcium Channels / metabolism*
  • Calcium Signaling
  • Calmodulin / metabolism*
  • Gene Knockout Techniques
  • HEK293 Cells
  • Humans
  • Hydrophobic and Hydrophilic Interactions
  • Membrane Proteins / metabolism
  • Models, Chemical
  • Models, Molecular
  • Mutation
  • Neoplasm Proteins / chemistry*
  • Neoplasm Proteins / genetics
  • Neoplasm Proteins / metabolism
  • Neoplasm Proteins / physiology*
  • ORAI1 Protein / chemistry
  • ORAI1 Protein / genetics
  • ORAI1 Protein / metabolism*
  • Protein Binding
  • Protein Domains
  • Stromal Interaction Molecule 1 / chemistry*
  • Stromal Interaction Molecule 1 / genetics
  • Stromal Interaction Molecule 1 / metabolism
  • Stromal Interaction Molecule 1 / physiology*
  • Up-Regulation

Substances

  • Calcium Channels
  • Calmodulin
  • Membrane Proteins
  • Neoplasm Proteins
  • ORAI1 Protein
  • ORAI1 protein, human
  • STIM1 protein, human
  • Stromal Interaction Molecule 1
  • Calcium