Gated regulation of CRAC channel ion selectivity by STIM1

Abstract

Two defining functional features of ion channels are ion selectivity and channel gating. Ion selectivity is generally considered an immutable property of the open channel structure, whereas gating involves transitions between open and closed channel states typically without changes in ion selectivity 1. In store-operated Ca2+ release-activated Ca2+ (CRAC) channels, the molecular mechanism of channel gating by the CRAC channel activator, STIM1 (stromal interaction molecule 1) remains unknown. CRAC channels are distinguished by an extraordinarily high Ca2+ selectivity and are instrumental in generating sustained [Ca2+]i elevations necessary for gene expression and effector function in many eukaryotic cells 2. Here, we probed the central features of the STIM1 gating mechanism in the CRAC channel protein, Orai1, and identified V102, a residue located in the extracellular region of the pore, as a candidate for the channel gate. Mutations at V102 produced constitutively active CRAC channels that were open even in the absence of STIM1. Unexpectedly, although STIM1-free V102 mutant channels were not Ca2+-selective, their Ca2+ selectivity was dose-dependently boosted by interactions with STIM1. Similar enhancement of Ca2+ selectivity also occurred in wild-type (WT) Orai1 channels by increasing the number of STIM1 activation domains directly tethered to Orai1 channels. Thus, exquisite Ca2+ selectivity is not an intrinsic property of CRAC channels, but rather a tunable feature bestowed on otherwise non-selective Orai1 channels by STIM1. Our results demonstrate that STIM1-mediated gating of CRAC channels occurs through an unusual mechanism wherein permeation and gating are closely coupled.

The STIM-Orai Pathway: Orai, the Pore-Forming Subunit of the CRAC Channel

“…In silico calculations for the Drosophila V174A channel—corresponding to a human V102A channel—suggest that it retains the closed pore conformation of the wildtype Drosophila channel but presents a markedly lower energetic barrier to ion conductance (Dong et al 2013). Consistent with the idea that the channel is not already in the STIM-gated conformation, the relatively nonselective V102X channels undergo a STIM-dependent conformational change to more selective channels (McNally et al 2012; Derler et al 2013). The Tb 3+ luminescence assay also detects a STIM-dependent conformational change in the V102A channel (Gudlur et al 2014).…”

“…A circumstantial case could be made that the SCID mutation blocks ion flux through an otherwise open channel in store-depleted cells. The R91W tryptophan ring is seen to occlude the channel in the Drosophila Orai crystal structure (Hou et al 2012), and experimentally the R91W replacement overrides the constitutive conductance of the V102C mutant (McNally et al 2012). However, both observations refer to the closed conformation of the channel.…”

“…Specifically, productive interaction with STIM1 leads to a rearrangement of the TM1 helices at E106 and at V102 in the nonpolar segment of the pore. The first direct evidence locating a gate in the ion-conducting path of Orai1 was the state-dependent accessibility of G98C to the covalent modifier MTSEA (McNally et al 2012). More recently, a gating movement of the wildtype channel has been observed directly by monitoring luminescence of Tb 3+ bound in the Orai1 Ca 2+ binding site (Gudlur et al 2014) (Fig.…”

“…Replacement of V102 by the small and more polar residues C, S, T, G, or A results in a constitutively conducting and less selective channel (McNally et al 2012; Derler et al 2013). In silico calculations for the Drosophila V174A channel—corresponding to a human V102A channel—suggest that it retains the closed pore conformation of the wildtype Drosophila channel but presents a markedly lower energetic barrier to ion conductance (Dong et al 2013).…”

Potent functional uncoupling between STIM1 and Orai1 by dimeric 2-aminodiphenyl borinate analogs

“…The Orai channel protein has four transmembrane helices and crystallography reveals the channel to be a hexamer [16]. The N-terminal helices of each of the six Orai subunits form the central Orai channel pore [17–20]. The Orai1 cytoplasmic N- and C-termini extend into the cytosol in close proximity to each other [16, 21].…”