Journal Article

STIM1 and calmodulin interact with Orai1 to induce Ca²⁺-dependent inactivation of CRAC channels

Dolmetsch, Ricardo E, Mullins, Franklin M, Chan, Young Park, Lewis, Richard S

2009

Overview

Ca²⁺-dependent inactivation (CDI) is a key regulator and hallmark of the Ca²⁺ release-activated Ca²⁺ (CRAC) channel, a prototypic store-operated Ca²⁺ channel. Although the roles of the endoplasmic reticulum Ca²⁺ sensor STIM1 and the channel subunit Orai1 in CRAC channel activation are becoming well understood, the molecular basis of CDI remains unclear. Recently, we defined a minimal CRAC activation domain (CAD; residues 342-448) that binds directly to Orai1 to activate the channel. Surprisingly, CAD-induced CRAC currents lack fast inactivation, revealing a critical role for STIM1 in this gating process. Through truncations of full-length STIM1, we identified a short domain (residues 470-491) C-terminal to CAD that is required for CDI. This domain contains a cluster of 7 acidic amino acids between residues 475 and 483. Neutralization of aspartate or glutamate pairs in this region either reduced or enhanced CDI, whereas the combined neutralization of six acidic residues eliminated inactivation entirely. Based on bioinformatics predictions of a calmodulin (CaM) binding site on Orai1, we also investigated a role for CaM in CDI. We identified a membrane-proximal N-terminal domain of Orai1 (residues 68-91) that binds CaM in a Ca²⁺-dependent manner and mutations that eliminate CaM binding abrogate CDI. These studies identify novel structural elements of STIM1 and Orai1 that are required for CDI and support a model in which CaM acts in concert with STIM1 and the N terminus of Orai1 to evoke rapid CRAC channel inactivation.

• Publisher: National Academy of Sciences,National Acad Sciences
• Subject: Amino acids / aspartic acid / Binding sites / bioinformatics / Biological Sciences / Calcium / calcium channels / Calcium Channels - metabolism / calmodulin / Calmodulin - metabolism / Cell Line / Cell membranes / Computational Biology / DNA Primers - genetics / DNA, Complementary - genetics / Electric current / Electrophysiology / endoplasmic reticulum / glutamic acid / HEK293 cells / Humans / Immunoblotting / Immunoprecipitation / Ion Channel Gating - physiology / Mast cells / Membrane Proteins - metabolism / Models, Biological / Mutagenesis / mutation / Neoplasm Proteins - metabolism / neutralization / ORAI1 Protein / Pipettes / Plasmids - genetics / prediction / Protein Binding / Protein Structure, Tertiary - genetics / Sensors / Stromal Interaction Molecule 1 / T lymphocytes / Transfection