Satanay Hubrack

Intramolecular shielding maintains the ER Ca2+ sensor STIM1 in an inactive conformation

Summary Store-operated calcium entry (SOCE) represents a major calcium influx pathway in non-excitable cells and is central to many physiological processes such as T cell activation and mast cell degranulation. SOCE is activated through intricate coordination between the Ca2+ sensor on the ER membrane (stromal interaction molecule 1, STIM1) and the plasma membrane channel Orai1. When Ca2+ stores are depleted, STIM1 oligomerizes and physically interacts with Orai1 through its SOAR/CAD domain, resulting in Orai1 gating and Ca2+ influx. Here, we describe novel inter- and intramolecular FRET sensors in the context of the full-length membrane-anchored STIM1, and show that STIM1 undergoes a conformational change in response to store depletion to adopt a stretched ‘open’ conformation that exposes SOAR/CAD and allows it to interact with Orai1. Mutational analyses reveal that electrostatic interactions between the predicted first and third coiled-coil domains of STIM1 are not involved in maintaining the ‘closed’ inactive conformation. In addition, the results argue that an amphipathic &agr;-helix between residues 317 and 336 in the so-called inhibitory domain is important to maintain STIM1 in a closed conformation at rest. Indeed, mutations that alter the amphipathic properties of this helix result in a STIM1 variant that is unable to respond to store depletion in terms of forming puncta, translocation to the cortical ER or activating Orai1.

Inositol 1,4,5-Trisphosphate (IP3) Receptor Up-regulation in Hypertension Is Associated with Sensitization of Ca2+ Release and Vascular Smooth Muscle Contractility

Background: The role of the vascular IP3 receptor (IP3R) in hypertension is unknown. Results: IP3R are up-regulated in vascular smooth muscle (VSM) in hypertension through the calcineurin-NFAT pathway. Conclusion: Up-regulated IP3R in VSM sensitize Ca2+ release and enhance contraction. Significance: Up-regulated vascular IP3R may contribute to vascular resistance in hypertension. Resistance arteries show accentuated responsiveness to vasoconstrictor agonists in hypertension, and this abnormality relies partly on enhanced Ca2+ signaling in vascular smooth muscle (VSM). Although inositol 1,4,5-triphosphate receptors (IP3Rs) are abundant in VSM, their role in the molecular remodeling of the Ca2+ signaling machinery during hypertension has not been addressed. Therefore, we compared IP3R expression and function between mesenteric arteries of normotensive and hypertensive animals. Levels of IP3R transcript and protein were significantly increased in mesenteric arteries of hypertensive animals, and pharmacological inhibition of the IP3R revealed a higher contribution of IP3-dependent Ca2+ release to vascular contraction in these arteries. Subsequently, we established cultured aortic VSM A7r5 cells as a cellular model that replicates IP3R up-regulation during hypertension by depolarizing the VSM cell membrane. IP3R up-regulation requires Ca2+ influx through L-type Ca2+ channels, followed by activation of the calcineurin-NFAT axis, resulting in IP3R transcription. Functionally, IP3R up-regulation in VSM is associated with enhancement and sensitization of IP3-dependent Ca2+ release, resulting in increased VSM contraction in response to agonist stimulation.

Endoplasmic Reticulum Remodeling Tunes IP3-Dependent Ca2+ Release Sensitivity

The activation of vertebrate development at fertilization relies on IP3-dependent Ca2+ release, a pathway that is sensitized during oocyte maturation. This sensitization has been shown to correlate with the remodeling of the endoplasmic reticulum into large ER patches, however the mechanisms involved are not clear. Here we show that IP3 receptors within ER patches have a higher sensitivity to IP3 than those in the neighboring reticular ER. The lateral diffusion rate of IP3 receptors in both ER domains is similar, and ER patches dynamically fuse with reticular ER, arguing that IP3 receptors exchange freely between the two ER compartments. These results suggest that increasing the density of IP3 receptors through ER remodeling is sufficient to sensitize IP3-dependent Ca2+ release. Mathematical modeling supports this concept of ‘geometric sensitization’ of IP3 receptors as a population, and argues that it depends on enhanced Ca2+-dependent cooperativity at sub-threshold IP3 concentrations. This represents a novel mechanism of tuning the sensitivity of IP3 receptors through ER remodeling during meiosis.

A STIM1-dependent ‘trafficking trap’ mechanism regulates Orai1 plasma membrane residence and Ca2+ influx levels

ABSTRACT The key proteins mediating store-operated Ca2+ entry (SOCE) are the endoplasmic reticulum (ER) Ca2+ sensor STIM1 and the plasma membrane Ca2+-selective channel Orai1. Here, we quantitatively dissect Orai1 trafficking dynamics and show that Orai1 recycles rapidly at the plasma membrane (Kex≃0.1 min−1), with ∼40% of the total Orai1 pool localizing to the plasma membrane at steady state. A subset of intracellular Orai1 localizes to a sub-plasmalemal compartment. Store depletion is coupled to Orai1 plasma membrane enrichment in a STIM1-dependent fashion. This is due to trapping of Orai1 into cortical ER STIM1 clusters, leading to its removal from the recycling pool and enrichment at the plasma membrane. Interestingly, upon high STIM1 expression, Orai1 is trapped into STIM1 clusters intracellularly, thus preventing its plasma membrane enrichment following store depletion. Consistent with this, STIM1 knockdown prevents trapping of excess Orai1 into limiting STIM1 clusters in the cortical ER. SOCE-dependent Ca2+ influx shows a similar biphasic dependence on the Orai1:STIM1 ratio. Therefore, a STIM1-dependent Orai1 ‘trafficking trap’ mechanism controls Orai1 plasma membrane enrichment and SOCE levels, thus modulating the SOCE ‘bandwidth’ for downstream signaling. Summary: Intracellular Orai1 trafficking to the plasma membrane in response to Ca2+ store depletion modulates store-operated Ca2+ entry influx through a ‘trafficking trap’ mechanism that depends on STIM1 expression levels.

The Xenopus TRPV6 homolog encodes a Mg2+-permeant channel that is inhibited by interaction with TRPC1

The TRP gene family encodes primarily cation non‐selective, Ca2+ permeant channels that are involved in a dizzying array of sensory mechanisms. Two channels in this large family TRPV5 and TRPV6 are highly Ca2+ selective and are expressed in epithelia where they are important in Ca2+ uptake. TRPV5/6 are constitutively active, yet the mechanisms regulating their activation in native tissue remains elusive. Here we functionally characterize the Xenopus TRPV6 homolog. xTRPV6 is expressed in the oocyte and encodes a channel that is permeant to divalents including Ca2+, and displays a high permeability to Mg2+. The oocyte does not exhibit functional TRPV6‐like current at rest, showing that the endogenous channel is somehow maintained in an inactive state. We show that endogenous as well as overexpressed xTRPV6 interacts with xTRPC1 and that this interaction inhibits xTRPV6 currents. As such TRPC1 is likely to regulate the activity of TRPV6 under physiological conditions. J. Cell. Physiol. 228: 2386–2398, 2013.

Phosphorylation of the rat Ins(1,4,5)P3 receptor at T930 within the coupling domain decreases its affinity to Ins(1,4,5)P3

The Ins(1,4,5)P3 receptor acts as a central hub for Ca2+ signaling by integrating multiple signaling modalities into Ca2+ release from intracellular stores downstream of G-protein and tyrosine kinase-coupled receptor stimulation. As such, the Ins(1,4,5)P3 receptor plays fundamental roles in cellular physiology. The regulation of the Ins(1,4,5)P3 receptor is complex and involves protein-protein interactions, post-translational modifications, allosteric modulation, and regulation of its sub-cellular distribution. Phosphorylation has been implicated in the sensitization of Ins(1,4,5)P3-dependent Ca2+ release observed during oocyte maturation. Here we investigate the role of phosphorylation at T-930, a residue phosphorylated specifically during meiosis. We show that a phosphomimetic mutation at T-930 of the rat Ins(1,4,5)P3 receptor results in decreased Ins(1,4,5)P3-dependent Ca2+ release and lowers the Ins(1,4,5)P3 binding affinity of the receptor. These data, coupled to the sensitization of Ins(1,4,5)P3-dependent Ca2+ release during meiosis, argue that phosphorylation within the coupling domain of the Ins(1,4,5)P3 receptor acts in a combinatorial fashion to regulate Ins(1,4,5)P3 receptor function.

The CCT chaperonin is a novel regulator of Ca2+ signaling through modulation of Orai1 trafficking

A chaperone complex (CCT) regulates the trafficking of a calcium channel (Orai1) and, as such, modulates cellular function. Store-operated Ca2+ entry (SOCE) encodes a range of cellular responses downstream of Ca2+ influx through the SOCE channel Orai1. Orai1 recycles at the plasma membrane (PM), with ~40% of the total Orai1 pool residing at the PM at steady state. The mechanisms regulating Orai1 recycling remain poorly understood. We map the domains in Orai1 that are required for its trafficking to and recycling at the PM. We further identify, using biochemical and proteomic approaches, the CCT [chaperonin-containing TCP-1 (T-complex protein 1)] chaperonin complex as a novel regulator of Orai1 recycling by primarily regulating Orai1 endocytosis. We show that Orai1 interacts with CCT through its intracellular loop and that inhibition of CCT-Orai1 interaction increases Orai1 PM residence. This increased residence is functionally significant as it results in prolonged Ca2+ signaling, early formation of STIM1-Orai1 puncta, and more rapid activation of NFAT (nuclear factor of activated T cells) downstream of SOCE. Therefore, the CCT chaperonin is a novel regulator of Orai1 trafficking and, as such, a modulator of Ca2+ signaling and effector activation kinetics.

Transition metal dependent regulation of the signal transduction cascade driving oocyte meiosis

The G2‐M transition of the cell cycle requires the activation of members of the Cdc25 dual specificity phosphatase family. Using Xenopus oocyte maturation as a model system, we have previously shown that chelation of transition metals blocks meiosis progression by inhibiting Cdc25C activation. Here, using approaches that allow for the isolation of very pure and active recombinant Cdc25C, we show that Cdc25C does not bind zinc as previously reported. Additionally, we show that mutants in the disordered C‐terminal end of Cdc25C are poor initiators of meiosis, likely due to their inability to localize to the proper sub‐cellular location. We further demonstrate that the transition metal chelator, TPEN, acts on or upstream of polo‐like kinases in the oocyte to block meiosis progression. Together our results provide novel insights into Cdc25C structure‐function relationship and the role of transition metals in regulating meiosis.