STIM1 and calmodulin interact with Orai1 to induce Ca ²⁺ -dependent inactivation of CRAC channels

Abstract

Ca 2؉ -dependent inactivation (CDI) is a key regulator and hallmark of the Ca 2؉ release-activated Ca 2؉ (CRAC) channel, a prototypic store-operated Ca 2؉ channel. Although the roles of the endoplasmic reticulum Ca 2؉ 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, CADinduced 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 Ca 2؉ -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.calcium ͉ ion channel gating ͉ store-operated calcium entry ͉ patch-clamp ͉ calcium-binding proteins S tore-operated Ca 2ϩ channels provide a major route for receptor-stimulated Ca 2ϩ entry in nonexcitable cells (1). The Ca 2ϩ -release activated Ca 2ϩ (CRAC) channel, the bestcharacterized store-operated channel, is essential for generating Ca 2ϩ signals that drive the activation of T lymphocytes, mast cells, and platelets (2-4). CRAC channel activity is shaped by the combination of store-dependent activation and Ca 2ϩ -dependent inactivation (CDI) processes. The mechanisms linking depletion of Ca 2ϩ from the endoplasmic reticulum (ER) to activation of the CRAC channel are becoming well understood: reduction of ER luminal Ca 2ϩ causes the ER Ca 2ϩ sensor STIM1 (5, 6) to oligomerize (7), enabling its accumulation at ER-plasma membrane junctions (3,8,9), where it binds directly to the CRAC channel subunit to open the channel (13,14).Compared with activation, much less is known about the mechanisms underlying CDI, one of the hallmark characteristics of the CRAC current (I CRAC ) in mammalian cells. Initial studies in mast cells and T cells revealed that Ca 2ϩ influx at hyperpolarized potentials drives rapid inactivation on a time scale of tens of milliseconds through the binding of Ca 2ϩ to sites located several nanometers from the intracellular mouth of the pore (15, 16). Subsequent studies have suggested that calmodulin (CaM) and STIM1 both may contribute to this process. In a rat liver cell l...

The inactivation domain of STIM1 is functionally coupled with the Orai1 pore to enable Ca2+-dependent inactivation

“…4). In previous studies (Derler et al, 2009b;Mullins et al, 2009), residual CDI was masked by potentiation, presumably because of low STIM1/Orai1 ratio (Scrimgeour et al, 2009;Hoover and Lewis, 2011). Our current results further underscore the importance of a high STIM1/Orai1 ratio for determining mutant CDI phenotypes.…”

“…Consistent with such a role, alanine or glycine substitutions for various combinations of acidic residues within this region reduced CDI, and full neutralization of all acidic residues eliminated CDI (Derler et al, 2009b;Lee et al, 2009;Mullins et al, 2009). However, an attempt to test whether the acidic region is the Ca 2+ sensor for CDI yielded equivocal results.…”

“…Neutralization of combinations of charged residues within ID STIM has been shown to eliminate, reduce, or enhance CDI, but mutations of individual residues have not been studied (Derler et al, 2009b;Lee et al, 2009;Mullins et al, 2009). To clarify the contributions of individual residues to CDI, we performed a full alanine scan of this negative charge cluster within ID STIM ( 475 DDVDDMDEE 483 ).…”

“…Consistent with such a role, alanine or glycine substitutions for various combinations of acidic residues within this region reduced CDI, and full neutralization of all acidic residues eliminated CDI (Derler et al, 2009b;Lee et al, 2009;Mullins et al, 2009 The inactivation domain of STIM1 (ID STIM : amino acids 470-491) has been described as necessary for Ca 2+ -dependent inactivation (CDI) of Ca 2+ release-activated Ca 2+ (CRAC) channels, but its mechanism of action is unknown. Here we identify acidic residues within ID STIM that control the extent of CDI and examine functional interactions of ID STIM with Orai1 pore residues W76 and Y80.…”

Orai1 pore residues control CRAC channel inactivation independently of calmodulin

“…STIM1 known as the inactivation domain of STIM (ID STIM , aa 470-491; Derler et al, 2009;Lee et al, 2009;Mullins et al, 2009), the intracellular II-III linker of Orai1 (aa 137-173; Srikanth et al, 2010), and the calmodulin (CaM)-binding domain of Orai1 (CBD Orai1 , aa 68-91; Mullins et al, 2009).…”