Beth A. McNally

Gated regulation of CRAC channel ion selectivity by STIM1

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. In store-operated Ca2+ release-activated Ca2+ (CRAC) channels, the molecular mechanism of channel gating by the CRAC channel activator, stromal interaction molecule 1 (STIM1), remains unknown. CRAC channels are distinguished by a very high Ca2+ selectivity and are instrumental in generating sustained intracellular calcium concentration elevations that are necessary for gene expression and effector function in many eukaryotic cells. Here we probe the central features of the STIM1 gating mechanism in the human CRAC channel protein, ORAI1, and identify V102, a residue located in the extracellular region of the pore, as a candidate for the channel gate. Mutations at V102 produce constitutively active CRAC channels that are open even in the absence of STIM1. Unexpectedly, although STIM1-free V102 mutant channels are not Ca2+-selective, their Ca2+ selectivity is dose-dependently boosted by interactions with STIM1. Similar enhancement of Ca2+ selectivity is also seen in wild-type ORAI1 channels by increasing the number of STIM1 activation domains that are directly tethered to ORAI1 channels, or by increasing the relative expression of full-length STIM1. Thus, exquisite Ca2+ selectivity is not an intrinsic property of CRAC channels but rather a tuneable feature that is bestowed on otherwise non-selective ORAI1 channels by STIM1. Our results demonstrate that STIM1-mediated gating of CRAC channels occurs through an unusual mechanism in which permeation and gating are closely coupled.

Structural determinants of ion permeation in CRAC channels

CRAC channels generate Ca2+ signals critical for the activation of immune cells and exhibit an intriguing pore profile distinguished by extremely high Ca2+ selectivity, low Cs+ permeability, and small unitary conductance. To identify the ion conduction pathway and gain insight into the structural bases of these permeation characteristics, we introduced cysteine residues in the CRAC channel pore subunit, Orai1, and probed their accessibility to various thiol-reactive reagents. Our results indicate that the architecture of the ion conduction pathway is characterized by a flexible outer vestibule formed by the TM1-TM2 loop, which leads to a narrow pore flanked by residues of a helical TM1 segment. Residues in TM3, and specifically, E190, a residue considered important for ion selectivity, are not close to the pore. Moreover, the outer vestibule does not significantly contribute to ion selectivity, implying that Ca2+ selectivity is conferred mainly by E106. The ion conduction pathway is sufficiently narrow along much of its length to permit stable coordination of Cd2+ by several TM1 residues, which likely explains the slow flux of ions within the restrained geometry of the pore. These results provide a structural framework to understand the unique permeation properties of CRAC channels.

Orai1 Mutations Alter Ion Permeation and Ca2+-dependent Fast Inactivation of CRAC Channels: Evidence for Coupling of Permeation and Gating

Ca2+ entry through store-operated Ca2+ release-activated Ca2+ (CRAC) channels is an essential trigger for lymphocyte activation and proliferation. The recent identification of Orai1 as a key CRAC channel pore subunit paves the way for understanding the molecular basis of Ca2+ selectivity, ion permeation, and regulation of CRAC channels. Previous Orai1 mutagenesis studies have indicated that a set of conserved acidic amino acids in trans membrane domains I and III and in the I–II loop (E106, E190, D110, D112, D114) are essential for the CRAC channels high Ca2+ selectivity. To further dissect the contribution of Orai1 domains important for ion permeation and channel gating, we examined the role of these conserved acidic residues on pore geometry, properties of Ca2+ block, and channel regulation by Ca2+. We find that alteration of the acidic residues lowers Ca2+ selectivity and results in striking increases in Cs+ permeation. This is likely the result of enlargement of the unusually narrow pore of the CRAC channel, thus relieving steric hindrance for Cs+ permeation. Ca2+ binding to the selectivity filter appears to be primarily affected by changes in the apparent on-rate, consistent with a rate-limiting barrier for Ca2+ binding. Unexpectedly, the mutations diminish Ca2+-mediated fast inactivation, a key mode of CRAC channel regulation. The decrease in fast inactivation in the mutant channels correlates with the decrease in Ca2+ selectivity, increase in Cs+ permeability, and enlargement of the pore. We propose that the structural elements involved in ion permeation overlap with those involved in the gating of CRAC channels.

The C‐ and N‐terminal STIM1 binding sites on Orai1 are required for both trapping and gating CRAC channels

•  The endoplasmic reticulum protein, stromal interaction molecule 1 (STIM1), activates Orai1 channels by directly interacting with each Orai1 subunit at the C‐ and N‐termini. Current models about the roles of these sites are rooted in notions of modularity, with the C‐terminal site thought to mediate STIM1 binding and the N‐terminal site thought to regulate channel gating. •  Here we report that the functions of the two sites are not so distinct: the N‐terminal site contributes to the stable association of STIM1 to Orai1, and, conversely, the C‐terminal site regulates channel activation. •  In addition to channel activation, STIM1 binding also modulates Orai1 channel ion selectivity. The structural requirements for modulation of ion selectivity closely match those seen for gating, suggesting that gating and permeation are closely coupled in Orai1 channels. •  These results help us understand the molecular requirements of STIM1‐mediated activation of Orai1 channels and regulation of channel ion selectivity.

Permeation, selectivity and gating in store‐operated CRAC channels

Abstract  Store‐operated Ca2+ release‐activated Ca2+ (CRAC) channels are a widespread mechanism for generating cellular Ca2+ signals and regulate many Ca2+‐dependent functions, including transcription, motility and proliferation. The opening of CRAC channels in response to depletion of intracellular Ca2+ stores involves a cascade of cellular events that culminate in direct interactions between STIM1, the endoplasmic reticulum Ca2+ sensor, and the channels composed of Orai proteins. Evidence gathered over the last two decades indicates that CRAC channels display a unique functional pore fingerprint characterized by exquisite Ca2+ selectivity, low unitary conductance, and low permeability to large cations. Here, we review the key pore properties of CRAC channels and discuss recent progress in addressing the molecular foundations of these properties. Structure–function and cysteine‐scanning studies have revealed the identity and organization of pore‐lining residues, including those that form the selectivity filter, providing a structural framework for understanding CRAC channel pore properties. Recent studies in pore mutants that produce STIM1‐independent constitutive channel activation indicate that exquisite Ca2+ selectivity in CRAC channels is not hardwired into Orai proteins, but is instead manifested only following the binding of STIM1 to the intrinsically poorly Ca2+‐selective Orai channels. These findings reveal new functional aspects of CRAC channels and suggest that the selectivity filter of the CRAC channel is a dynamic structure whose conformation and functional properties are powerfully regulated by the channel activation stimulus.

Targeting lymphotoxin-mediated negative selection to prevent prostate cancer in mice with genetic predisposition

The identification of individuals genetically susceptible to cancer calls for preventive measures to minimize the cancer risk in these high-risk populations. Immune prevention is made necessary by the anticipated health threat, but lack of enough high-affinity T cells against tumor-associated antigens and the unpredictability of tumor antigens make antigen-based immune prevention untenable for cancer. To address this issue, we explored a non-antigen-based cancer immune prevention strategy using the transgenic adenocarcinoma of mouse prostate model that spontaneously develops prostate cancer with 100% penetrance. We show that targeted mutation of the lymphotoxin α (LTα) gene efficiently rescued tumor-reactive T cells, drastically reduced cancer incidence, and almost completely ablated metastasis. Remarkably, short-term treatments with the fusion protein consisting of constant region of IgG and extracellular domain of lymphotoxin β receptor (LTβRIg) interrupted clonal deletion, reduced the size of the primary cancer, and completely prevented metastasis later in life. Our data demonstrated the value of non-antigen-based immune prevention for those with a genetic predisposition to cancer.

STIM1 activates CRAC channels through rotation of the pore helix to open a hydrophobic gate

Store-operated Ca2+ release-activated Ca2+ (CRAC) channels constitute a major pathway for Ca2+ influx and mediate many essential signalling functions in animal cells, yet how they open remains elusive. Here, we investigate the gating mechanism of the human CRAC channel Orai1 by its activator, stromal interacting molecule 1 (STIM1). We find that two rings of pore-lining residues, V102 and F99, work together to form a hydrophobic gate. Mutations of these residues to polar amino acids produce channels with leaky gates that conduct ions in the resting state. STIM1-mediated channel activation occurs through rotation of the pore helix, which displaces the F99 residues away from the pore axis to increase pore hydration, allowing ions to flow through the V102-F99 hydrophobic band. Pore helix rotation by STIM1 also explains the dynamic coupling between CRAC channel gating and ion selectivity. This hydrophobic gating mechanism has implications for CRAC channel function, pharmacology and disease-causing mutations.

Structural Determinants of Ion Permeation in Crac Channels

CRAC channels generate Ca2+ signals critical for the activation of immune cells and exhibit an intriguing pore profile distinguished by extremely high Ca2+ selectivity, low Cs+ permeability, and small unitary conductance. To identify the conduction pathway of the transported ions and gain insight into the structural bases of these characteristics, we introduced cysteine residues in the CRAC channel pore subunit, Orai1, and probed their accessibility to various thiol-reactive reagents. Our results indicate that the architecture of the ion conduction pathway is characterized by a flexible outer vestibule formed by the TM1-TM2 loop, which leads to a narrow pore flanked by residues of a helical TM1 segment. Residues in TM3, and specifically, E190, a residue considered important for ion selectivity, are not close to the pore. Moreover, the outer vestibule does not significantly contribute to ion selectivity, implying that Ca2+ selectivity is conferred mainly by E106 in the TM1 segment. The pore is sufficiently narrow along much of its length to permit stable coordination of Cd2+ by several TM1 residues, which likely explains the slow flux of ions within the restrained geometry of the pore. Together, these results reveal new insights into the long-sought structural basis for the unique permeation properties of CRAC channels.