α-SNAP regulates dynamic, on-site assembly and calcium selectivity of Orai1 channels

Li, Peiyao; Miao, Yong; Dani, Adish; Vig, Monika

doi:10.1091/mbc.e16-03-0163

Cited by 24 publications

(40 citation statements)

References 55 publications

(97 reference statements)

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“…Such an assembly is consistent with other reports that indicate Orai channels are assembled from dimers (29,33). Indeed, recent data indicate that PM dimers predominate in the resting state (90%) and can assemble into multimers up to hexamers, upon store depletion (34). This lends support to the evidence presented here that several combinations of concatemers may form hexamers through the assembly of dimers.…”

Section: Journal Of Biological Chemistrysupporting

confidence: 93%

“…The lack of function of the XO and OX dimers indicates that functional channels are not formed from the assembly of six dimers each contributing just one functional monomer to the hexameric assembly. Thus, we conclude that the Orai channel may be assembled from dimers, in agreement with other studies (29,33,34) as well as with the crystallographic structure that reveals that Orai channels comprise a trimer-of-dimers (22).…”

Section: A Model For the Functional Assembly Of Orai1supporting

confidence: 92%

“…Shortly after, a number of other studies using a variety of approaches, including recovery after photobleaching, cross-linking, and electron microscopy, reported a similar tetrameric subunit structure for Orai channels (29 -33). Reports have also suggested that Orai channels may exist as dimers, which could associate into tetramers (29,33) or possibly hexamers (34). A seemingly more definitive study from Thompson and Shuttleworth (23) directly compared the functional channel properties of tetrameric and hexameric concatemers of Orai1.…”

Section: Entry Moreover Each Orai1 Concatemer Mediated Camentioning

confidence: 99%

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The Orai1 Store-operated Calcium Channel Functions as a Hexamer

Cai

Zhou

Nwokonko

et al. 2016

Journal of Biological Chemistry

102

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Edited by Roger ColbranOrai channels mediate store-operated Ca 2؉ signals crucial in regulating transcription in many cell types, and implicated in numerous immunological and inflammatory disorders. Despite their central importance, controversy surrounds the basic subunit structure of Orai channels, with several biochemical and biophysical studies suggesting a tetrameric structure yet crystallographic evidence indicating a hexamer. We systematically investigated the subunit configuration of the functional Orai1 channel, generating a series of tdTomato-tagged concatenated Orai1 channel constructs (dimers to hexamers) expressed in CRISPR-derived ORAI1 knock-out HEK cells, stably expressing STIM1-YFP. Surface biotinylation demonstrated that the fulllength concatemers were surface membrane-expressed. Unexpectedly, Orai1 dimers, trimers, tetramers, pentamers, and hexamers all mediated similar and substantial store-operated Ca 2؉ entry. Moreover, each Orai1 concatemer mediated Ca 2؉ currents with inward rectification and reversal potentials almost identical to those observed with expressed Orai1 monomer. In Orai1 tetramers, subunit-specific replacement with Orai1 E106A "pore-inactive" subunits revealed that functional channels utilize only the N-terminal dimer from the tetramer. In contrast, Orai1 E106A replacement in Orai1 hexamers established that all the subunits can contribute to channel formation, indicating a hexameric channel configuration. The critical Ca 2؉ selectivity filter-forming Glu-106 residue may mediate Orai1 channel assembly around a central Ca 2؉ ion within the pore. Thus, multiple E106A substitutions in the Orai1 hexamer may promote an alternative "trimer-of-dimers" channel configuration in which the C-terminal E106A subunits are excluded from the hexameric core. Our results argue strongly against a tetrameric configuration for Orai1 channels and indicate that the Orai1 channel functions as a hexamer.

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Section: Journal Of Biological Chemistrysupporting

confidence: 93%

Section: A Model For the Functional Assembly Of Orai1supporting

confidence: 92%

Section: Entry Moreover Each Orai1 Concatemer Mediated Camentioning

confidence: 99%

See 1 more Smart Citation

The Orai1 Store-operated Calcium Channel Functions as a Hexamer

Cai

Zhou

Nwokonko

et al. 2016

Journal of Biological Chemistry

102

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“…Loss of accessory factors might well explain NAADP insensitivity in some studies. Indeed, such loss might also affect the ion selectivity of TPCs as accessory proteins are known to dictate the ion selectivity of other channels such as Orai [77] and MCU [78].…”

Section: Discussionmentioning

confidence: 99%

Mining of Ebola virus entry inhibitors identifies approved drugs as two-pore channel pore blockers

Penny

Vassileva

Jha

et al. 2019

Biochimica et Biophysica Acta (BBA) - Molecular Cell Research

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A B S T R A C TTwo-pore channels (TPCs) are Ca 2+ -permeable ion channels localised to the endo-lysosomal system where they regulate trafficking of various cargoes including viruses. As a result, TPCs are emerging as important drug targets. However, their pharmacology is ill-defined. There are no approved drugs to target them. And their mechanism of ligand activation is largely unknown. Here, we identify a number of FDA-approved drugs as TPC pore blockers. Using a model of the pore of human TPC2 based on recent structures of mammalian TPCs, we virtually screened a database of~1500 approved drugs. Because TPCs have recently emerged as novel host factors for Ebola virus entry, we reasoned that Ebola virus entry inhibitors may exert their effects through inhibition of TPCs. Cross-referencing hits from the TPC virtual screen with two recent high throughput anti-Ebola screens yielded approved drugs targeting dopamine and estrogen receptors as common hits. These compounds inhibited endogenous NAADP-evoked Ca 2+ release from sea urchin egg homogenates, NAADP-mediated channel activity of TPC2 re-routed to the plasma membrane, and PI(3,5)P 2 -mediated channel activity of TPC2 expressed in enlarged lysosomes. Mechanistically, single channel analyses showed that the drugs reduced mean open time consistent with a direct action on the pore. Functionally, drug potency in blocking TPC2 activity correlated with inhibition of Ebola virus-like particle entry. Our results expand TPC pharmacology through the identification of approved drugs as novel blockers, support a role for TPCs in Ebola virus entry, and provide insight into the mechanisms underlying channel regulation.

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“…The background was calculated locally from a 1 pixel thick frame 238 around the region and subtracted from the signal. Spots with signal to noise ratios less than 2, 239 those that were fluorescent in the first frame of the image sequence and spots that lasted less 240 than five frames were excluded from the analysis as we couldn't reliably count the number of 241 steps from their intensity-time traces (Li et al, 2016). The steps in the traces were counted 242 manually.…”

Section: Counting Of the Number Of Glun2a Subunits In Exocytic Vesiclmentioning

confidence: 99%

Long-Term Potentiation Induces Extrasynaptic Exocytosis of Glun2A-containing NMDA Receptors that is Mainly Controlled by SNAP23

Li²,

Wang

2019

Preprint

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NMDA receptors (NMDAR) are key players in the initiation of synaptic plasticity that underlies learning and memory. Long-term potentiation (LTP) of synapses require an increased calcium current via NMDA channels to trigger modifications in postsynaptic density (PSD). It is generally believed that the amount of NMDARs on the postsynaptic surface remains stationary, whereas their subunit composition is dynamically fluctuated during this plasticity process. However, the molecular machinery underlying this subunit-specific regulation remains largely elusive. Here, by detecting the time-lapse changes of surface GluN2A and GluN2B subunit levels using biochemical approaches, surface immunostaining, live-imaging and super-resolution microscopy, we uncovered a transient increase of surface GluN2A-type NMDARs shortly after the induction of chemical long term potentiation (cLTP). These augmented sub-diffraction-limited GluN2A clusters predominantly exist in extrasynaptic domains. We also showed that the spine-enriched SNARE associated protein SNAP-23, and to a minor extent its homologue SNAP-25, control both the basal and regulated surface level of GluN2A receptors. Using a total internal reflection fluorescence microscopy (TIRFM) based live-imaging assay, we resolved and analyzed individual exocytic events of NMDARs in live neurons and found that cLTP raised the frequency of NMDAR exocytosis at extrasynaptic regions, without altering the duration or the package size of these events. Our study thereby provides direct evidence that synaptic plasticity controls the postsynaptic exocytosis machinery, which induces the insertion of more GluN2A receptors into the extrasynaptic area.Significance StatementMemory formation involves the long-term modification of synapses, which is called synaptic plasticity. In the postsynaptic density (PSD) of excited neurons, this modification process occurs on a minute timescale, initiated by the opening of NMDARs that trigger downstream cascades to fix the potentiation (LTP) at specific synapses for longer timescales. Here, using a novel live-imaging assay we resolved the dynamic delivery of NMDARs to the cell surface, and found that only the insertion frequency, not the duration of individual insertion or number of GluN2A subunits each of these NMDAR vesicles contains, was altered during the synaptic potentiation process. We also identified SNAP-23 as the key molecule mediating this activity dependent NMDAR surface delivery. This study provides a novel mechanism of how NMDARs are regulated in the short window to initiate the long-lasting synaptic modifications.

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α-SNAP regulates dynamic, on-site assembly and calcium selectivity of Orai1 channels

Cited by 24 publications

References 55 publications

The Orai1 Store-operated Calcium Channel Functions as a Hexamer

The Orai1 Store-operated Calcium Channel Functions as a Hexamer

Mining of Ebola virus entry inhibitors identifies approved drugs as two-pore channel pore blockers

Long-Term Potentiation Induces Extrasynaptic Exocytosis of Glun2A-containing NMDA Receptors that is Mainly Controlled by SNAP23

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