Cathepsin S Causes Inflammatory Pain via Biased Agonism of PAR2 and TRPV4

Serine proteases such as trypsin and mast cell tryptase cleave protease-activated receptor-2 (PAR₂) at R³⁶↓S³⁷ and reveal a tethered ligand that excites nociceptors, causing neurogenic inflammation and pain. Whether proteases that cleave PAR₂ at distinct sites are biased agonists that also induce inflammation and pain is unexplored. Cathepsin S (Cat-S) is a lysosomal cysteine protease of antigen-presenting cells that is secreted during inflammation and which retains activity at extracellular pH. We observed that Cat-S cleaved PAR₂ at E⁵⁶↓T⁵⁷, which removed the canonical tethered ligand and prevented trypsin activation. In HEK and KNRK cell lines and in nociceptive neurons of mouse dorsal root ganglia, Cat-S and a decapeptide mimicking the Cat-S-revealed tethered ligand-stimulated PAR₂ coupling to Gαs and formation of cAMP. In contrast to trypsin, Cat-S did not mobilize intracellular Ca²⁺, activate ERK1/2, recruit β-arrestins, or induce PAR₂ endocytosis. Cat-S caused PAR₂-dependent activation of transient receptor potential vanilloid 4 (TRPV4) in Xenopus laevis oocytes, HEK cells and nociceptive neurons, and stimulated neuronal hyperexcitability by adenylyl cyclase and protein kinase A-dependent mechanisms. Intraplantar injection of Cat-S caused inflammation and hyperalgesia in mice that was attenuated by PAR₂ or TRPV4 deletion and adenylyl cyclase inhibition. Cat-S and PAR₂ antagonists suppressed formalin-induced inflammation and pain, which implicates endogenous Cat-S and PAR₂ in inflammatory pain. Our results identify Cat-S as a biased agonist of PAR₂ that causes PAR₂- and TRPV4-dependent inflammation and pain. They expand the role of PAR₂ as a mediator of protease-driven inflammatory pain.     

Ca2+ Signals Generated by CatSper and Ca2+ Stores Regulate Different Behaviors in Human Sperm

[Ca²⁺]_i signaling regulates sperm motility, enabling switching between functionally different behaviors that the sperm must employ as it ascends the female tract and fertilizes the oocyte. We report that different behaviors in human sperm are recruited according to the Ca²⁺ signaling pathway used. Activation of CatSper (by raising pH_i or stimulating with progesterone) caused sustained [Ca²⁺]_i elevation but did not induce hyperactivation, the whiplash-like behavior required for progression along the oviduct and penetration of the zona pellucida. In contrast, penetration into methylcellulose (mimicking penetration into cervical mucus or cumulus matrix) was enhanced by activation of CatSper. NNC55-0396, which abolishes CatSper currents in human sperm, inhibited this effect. Treatment with 5 μm thimerosal to mobilize stored Ca²⁺ caused sustained [Ca²⁺]_i elevation and induced strong, sustained hyperactivation that was completely insensitive to NNC55-0396. Thimerosal had no effect on penetration into methylcellulose. 4-Aminopyridine, a powerful modulator of sperm motility, both raised pH_i and mobilized Ca²⁺ stored in sperm (and from microsomal membrane preparations). 4-Aminopyridine-induced hyperactivation even in cells suspended in Ca²⁺-depleted medium and also potentiated penetration into methylcellulose. The latter effect was sensitive to NNC55-039, but induction of hyperactivation was not. We conclude that these two components of the [Ca²⁺]_i signaling apparatus have strikingly different effects on sperm motility. Furthermore, since stored Ca²⁺ at the sperm neck can be mobilized by Ca²⁺-induced Ca²⁺ release, we propose that CatSper activation can elicit functionally different behaviors according to the sensitivity of the Ca²⁺ store, which may be regulated by capacitation and NO from the cumulus.     

Protease-activated Receptor 2 (PAR2) Protein and Transient Receptor Potential Vanilloid 4 (TRPV4) Protein Coupling Is Required for Sustained Inflammatory Signaling

G protein-coupled receptors of nociceptive neurons can sensitize transient receptor potential (TRP) ion channels, which amplify neurogenic inflammation and pain. Protease-activated receptor 2 (PAR₂), a receptor for inflammatory proteases, is a major mediator of neurogenic inflammation and pain. We investigated the signaling mechanisms by which PAR₂ regulates TRPV4 and determined the importance of tyrosine phosphorylation in this process. Human TRPV4 was expressed in HEK293 cells under control of a tetracycline-inducible promoter, allowing controlled and graded channel expression. In cells lacking TRPV4, the PAR₂ agonist stimulated a transient increase in [Ca²⁺]_i. TRPV4 expression led to a markedly sustained increase in [Ca²⁺]_i. Removal of extracellular Ca²⁺ and treatment with the TRPV4 antagonists Ruthenium Red or HC067047 prevented the sustained response. Inhibitors of phospholipase A₂ and cytochrome P450 epoxygenase attenuated the sustained response, suggesting that PAR₂ generates arachidonic acid-derived lipid mediators, such as 5′,6′-EET, that activate TRPV4. Src inhibitor 1 suppressed PAR₂-induced activation of TRPV4, indicating the importance of tyrosine phosphorylation. The TRPV4 tyrosine mutants Y110F, Y805F, and Y110F/Y805F were expressed normally at the cell surface. However, PAR₂ was unable to activate TRPV4 with the Y110F mutation. TRPV4 antagonism suppressed PAR₂ signaling to primary nociceptive neurons, and TRPV4 deletion attenuated PAR₂-stimulated neurogenic inflammation. Thus, PAR₂ activation generates a signal that induces sustained activation of TRPV4, which requires a key tyrosine residue (TRPV4-Tyr-110). This mechanism partly mediates the proinflammatory actions of PAR₂.     

Neutrophil Elastase Activates Protease-activated Receptor-2 (PAR2) and Transient Receptor Potential Vanilloid 4 (TRPV4) to Cause Inflammation and Pain

Proteases that cleave protease-activated receptor-2 (PAR₂) at Arg³⁶↓Ser³⁷ reveal a tethered ligand that binds to the cleaved receptor. PAR₂ activates transient receptor potential (TRP) channels of nociceptive neurons to induce neurogenic inflammation and pain. Although proteases that cleave PAR₂ at non-canonical sites can trigger distinct signaling cascades, the functional importance of the PAR₂-biased agonism is uncertain. We investigated whether neutrophil elastase, a biased agonist of PAR₂, causes inflammation and pain by activating PAR₂ and TRP vanilloid 4 (TRPV4). Elastase cleaved human PAR₂ at Ala⁶⁶↓Ser⁶⁷ and Ser⁶⁷↓Val⁶⁸_. Elastase stimulated PAR₂-dependent cAMP accumulation and ERK1/2 activation, but not Ca²⁺ mobilization, in KNRK cells. Elastase induced PAR₂ coupling to Gα_s but not Gα_q in HEK293 cells. Although elastase did not promote recruitment of G protein-coupled receptor kinase-2 (GRK2) or β-arrestin to PAR₂, consistent with its inability to promote receptor endocytosis, elastase did stimulate GRK6 recruitment. Elastase caused PAR₂-dependent sensitization of TRPV4 currents in Xenopus laevis oocytes by adenylyl cyclase- and protein kinase A (PKA)-dependent mechanisms. Elastase stimulated PAR₂-dependent cAMP formation and ERK1/2 phosphorylation, and a PAR₂- and TRPV4-mediated influx of extracellular Ca²⁺ in mouse nociceptors. Adenylyl cyclase and PKA-mediated elastase-induced activation of TRPV4 and hyperexcitability of nociceptors. Intraplantar injection of elastase to mice caused edema and mechanical hyperalgesia by PAR₂- and TRPV4-mediated mechanisms. Thus, the elastase-biased agonism of PAR₂ causes Gα_s-dependent activation of adenylyl cyclase and PKA, which activates TRPV4 and sensitizes nociceptors to cause inflammation and pain. Our results identify a novel mechanism of elastase-induced activation of TRPV4 and expand the role of PAR₂ as a mediator of protease-driven inflammation and pain.     

Modulation of Endoplasmic Reticulum Ca2+ Store Filling by Cyclic ADP-ribose Promotes Inositol Trisphosphate (IP3)-evoked Ca2+ Signals

In addition to its well established function in activating Ca²⁺ release from the endoplasmic reticulum (ER) through ryanodine receptors (RyR), the second messenger cyclic ADP-ribose (cADPR) also accelerates the activity of SERCA pumps, which sequester Ca²⁺ into the ER. Here, we demonstrate a potential physiological role for cADPR in modulating cellular Ca²⁺ signals via changes in ER Ca²⁺ store content, by imaging Ca²⁺ liberation through inositol trisphosphate receptors (IP₃R) in Xenopus oocytes, which lack RyR. Oocytes were injected with the non-metabolizable analog 3-deaza-cADPR, and cytosolic [Ca²⁺] was transiently elevated by applying voltage-clamp pulses to induce Ca²⁺ influx through expressed plasmalemmal nicotinic channels. We observed a subsequent potentiation of global Ca²⁺ signals evoked by strong photorelease of IP₃, and increased numbers of local Ca²⁺ puffs evoked by weaker photorelease. These effects were not evident with cADPR alone or following cytosolic Ca²⁺ elevation alone, indicating that they did not arise through direct actions of cADPR or Ca²⁺ on the IP₃R, but likely resulted from enhanced ER store filling. Moreover, the appearance of a new population of puffs with longer latencies, prolonged durations, and attenuated amplitudes suggests that luminal ER Ca²⁺ may modulate IP₃R function, in addition to simply determining the size of the available store and the electrochemical driving force for release.     

Identification of an l-Phenylalanine Binding Site Enhancing the Cooperative Responses of the Calcium-sensing Receptor to Calcium

Functional positive cooperative activation of the extracellular calcium ([Ca²⁺]_o)-sensing receptor (CaSR), a member of the family C G protein-coupled receptors, by [Ca²⁺]_o or amino acids elicits intracellular Ca²⁺ ([Ca²⁺]_i) oscillations. Here, we report the central role of predicted Ca²⁺-binding site 1 within the hinge region of the extracellular domain (ECD) of CaSR and its interaction with other Ca²⁺-binding sites within the ECD in tuning functional positive homotropic cooperativity caused by changes in [Ca²⁺]_o. Next, we identify an adjacent l-Phe-binding pocket that is responsible for positive heterotropic cooperativity between [Ca²⁺]_o and l-Phe in eliciting CaSR-mediated [Ca²⁺]_i oscillations. The heterocommunication between Ca²⁺ and an amino acid globally enhances functional positive homotropic cooperative activation of CaSR in response to [Ca²⁺]_o signaling by positively impacting multiple [Ca²⁺]_o-binding sites within the ECD. Elucidation of the underlying mechanism provides important insights into the longstanding question of how the receptor transduces signals initiated by [Ca²⁺]_o and amino acids into intracellular signaling events.     

Calcium-induced conformational changes in C-terminal tail of polycystin-2 are necessary for channel gating

Polycystin-2 (PC2) is a Ca²⁺-permeable transient receptor potential channel activated and regulated by changes in cytoplasmic Ca²⁺. PC2 mutations are responsible for ∼15% of autosomal dominant polycystic kidney disease. Although the C-terminal cytoplasmic tail of PC2 has been shown to contain a Ca²⁺-binding EF-hand domain, the molecular basis of PC2 channel gating by Ca²⁺ remains unknown. We propose that the PC2 EF-hand is a Ca²⁺ sensor required for channel gating. Consistent with this, Ca²⁺ binding causes a dramatic decrease in the radius of gyration (R_g) of the PC2 EF-hand by small angle x-ray scattering and significant conformational changes by NMR. Furthermore, increasing Ca²⁺ concentrations cause the C-terminal cytoplasmic tail to transition from a mixture of extended oligomers to a single compact dimer by analytical ultracentrifugation, coupled with a >30 Å decrease in maximum interatomic distance (D_max) by small angle x-ray scattering. Mutant PC2 channels unable to bind Ca²⁺ via the EF-hand are inactive in single-channel planar lipid bilayers and inhibit Ca²⁺ release from ER stores upon overexpression in cells, suggesting dominant negative properties. Our results support a model where PC2 channels are gated by discrete conformational changes in the C-terminal cytoplasmic tail in response to changes in cytoplasmic Ca²⁺ levels. These properties of PC2 are lost in autosomal dominant polycystic kidney disease, emphasizing the importance of PC2 to kidney cell function. We speculate that PC2 and the Ca²⁺-dependent transient receptor potential channels in general are regulated by similar conformational changes in their cytoplasmic domains that are propagated to the channel pore.     

Ca2+ Influx via the Na+/Ca2+ Exchanger Is Enhanced in Malignant Hyperthermia Skeletal Muscle

Malignant hyperthermia (MH) is potentially fatal pharmacogenetic disorder of skeletal muscle caused by intracellular Ca²⁺ dysregulation. NCX is a bidirectional transporter that effluxes (forward mode) or influxes (reverse mode) Ca²⁺ depending on cellular activity. Resting intracellular calcium ([Ca²⁺]_r) and sodium ([Na⁺]_r) concentrations are elevated in MH susceptible (MHS) swine and murine muscles compared with their normal (MHN) counterparts, although the contribution of NCX is unclear. Lowering [Na⁺]_e elevates [Ca²⁺]_r in both MHN and MHS swine muscle fibers and it is prevented by removal of extracellular Ca²⁺ or reduced by t-tubule disruption, in both genotypes. KB-R7943, a nonselective NCX3 blocker, reduced [Ca²⁺]_r in both swine and murine MHN and MHS muscle fibers at rest and decreased the magnitude of the elevation of [Ca²⁺]_r observed in MHS fibers after exposure to halothane. YM-244769, a high affinity reverse mode NCX3 blocker, reduces [Ca²⁺]_r in MHS muscle fibers and decreases the amplitude of [Ca²⁺]_r rise triggered by halothane, but had no effect on [Ca²⁺]_r in MHN muscle. In addition, YM-244769 reduced the peak and area under the curve of the Ca²⁺ transient elicited by high [K⁺]_e and increased its rate of decay in MHS muscle fibers. siRNA knockdown of NCX3 in MHS myotubes reduced [Ca²⁺]_r and the Ca²⁺ transient area induced by high [K⁺]_e. These results demonstrate a functional NCX3 in skeletal muscle whose activity is enhanced in MHS. Moreover reverse mode NCX3 contributes to the Ca²⁺ transients associated with K⁺-induced depolarization and the halothane-triggered MH episode in MHS muscle fibers.     

A Reciprocal Shift in Transient Receptor Potential Channel 1 (TRPC1) and Stromal Interaction Molecule 2 (STIM2) Contributes to Ca2+ Remodeling and Cancer Hallmarks in Colorectal Carcinoma Cells

We have investigated the molecular basis of intracellular Ca²⁺ handling in human colon carcinoma cells (HT29) versus normal human mucosa cells (NCM460) and its contribution to cancer features. We found that Ca²⁺ stores in colon carcinoma cells are partially depleted relative to normal cells. However, resting Ca²⁺ levels, agonist-induced Ca²⁺ increases, store-operated Ca²⁺ entry (SOCE), and store-operated currents (I_SOC) are largely enhanced in tumor cells. Enhanced SOCE and depleted Ca²⁺ stores correlate with increased cell proliferation, invasion, and survival characteristic of tumor cells. Normal mucosa cells displayed small, inward Ca²⁺ release-activated Ca²⁺ currents (I_CRAC) mediated by ORAI1. In contrast, colon carcinoma cells showed mixed currents composed of enhanced I_CRAC plus a nonselective I_SOC mediated by TRPC1. Tumor cells display increased expression of TRPC1, ORAI1, ORAI2, ORAI3, and STIM1. In contrast, STIM2 protein was nearly depleted in tumor cells. Silencing data suggest that enhanced ORAI1 and TRPC1 contribute to enhanced SOCE and differential store-operated currents in tumor cells, whereas ORAI2 and -3 are seemingly less important. In addition, STIM2 knockdown decreases SOCE and Ca²⁺ store content in normal cells while promoting apoptosis resistance. These data suggest that loss of STIM2 may underlie Ca²⁺ store depletion and apoptosis resistance in tumor cells. We conclude that a reciprocal shift in TRPC1 and STIM2 contributes to Ca²⁺ remodeling and tumor features in colon cancer.     

Calmodulin Regulates Ca2+-sensing Receptor-mediated Ca2+ Signaling and Its Cell Surface Expression

The Ca²⁺-sensing receptor (CaSR) is a member of family C of the GPCRs responsible for sensing extracellular Ca²⁺ ([Ca²⁺]_o) levels, maintaining extracellular Ca²⁺ homeostasis, and transducing Ca²⁺ signaling from the extracellular milieu to the intracellular environment. In the present study, we have demonstrated a Ca²⁺-dependent, stoichiometric interaction between CaM and a CaM-binding domain (CaMBD) located within the C terminus of CaSR (residues 871–898). Our studies suggest a wrapping around 1–14-like mode of interaction that involves global conformational changes in both lobes of CaM with concomitant formation of a helical structure in the CaMBD. More importantly, the Ca²⁺-dependent association between CaM and the C terminus of CaSR is critical for maintaining proper responsiveness of intracellular Ca²⁺ responses to changes in extracellular Ca²⁺ and regulating cell surface expression of the receptor.     

Ca2+ Sparks Act as Potent Regulators of Excitation-Contraction Coupling in Airway Smooth Muscle

Ca²⁺ sparks are short lived and localized Ca²⁺ transients resulting from the opening of ryanodine receptors in sarcoplasmic reticulum. These events relax certain types of smooth muscle by activating big conductance Ca²⁺-activated K⁺ channels to produce spontaneous transient outward currents (STOCs) and the resultant closure of voltage-dependent Ca²⁺ channels. But in many smooth muscles from a variety of organs, Ca²⁺ sparks can additionally activate Ca²⁺-activated Cl⁻ channels to generate spontaneous transient inward current (STICs). To date, the physiological roles of Ca²⁺ sparks in this latter group of smooth muscle remain elusive. Here, we show that in airway smooth muscle, Ca²⁺ sparks under physiological conditions, activating STOCs and STICs, induce biphasic membrane potential transients (BiMPTs), leading to membrane potential oscillations. Paradoxically, BiMPTs stabilize the membrane potential by clamping it within a negative range and prevent the generation of action potentials. Moreover, blocking either Ca²⁺ sparks or hyperpolarization components of BiMPTs activates voltage-dependent Ca²⁺ channels, resulting in an increase in global [Ca²⁺]_i and cell contraction. Therefore, Ca²⁺ sparks in smooth muscle presenting both STICs and STOCs act as a stabilizer of membrane potential, and altering the balance can profoundly alter the status of excitability and contractility. These results reveal a novel mechanism underlying the control of excitability and contractility in smooth muscle.     

Elucidation of a Novel Extracellular Calcium-binding Site on Metabotropic Glutamate Receptor 1α (mGluR1α) That Controls Receptor Activation

Metabotropic glutamate receptor 1α (mGluR1α) exerts important effects on numerous neurological processes. Although mGluR1α is known to respond to extracellular Ca²⁺ ([Ca²⁺]_o) and the crystal structures of the extracellular domains (ECDs) of several mGluRs have been determined, the calcium-binding site(s) and structural determinants of Ca²⁺-modulated signaling in the Glu receptor family remain elusive. Here, we identify a novel Ca²⁺-binding site in the mGluR1α ECD using a recently developed computational algorithm. This predicted site (comprising Asp-318, Glu-325, and Asp-322 and the carboxylate side chain of the receptor agonist, Glu) is situated in the hinge region in the ECD of mGluR1α adjacent to the reported Glu-binding site, with Asp-318 involved in both Glu and calcium binding. Mutagenesis studies indicated that binding of Glu and Ca²⁺ to their distinct but partially overlapping binding sites synergistically modulated mGluR1α activation of intracellular Ca²⁺ ([Ca²⁺]_i) signaling. Mutating the Glu-binding site completely abolished Glu signaling while leaving its Ca²⁺-sensing capability largely intact. Mutating the predicted Ca²⁺-binding residues abolished or significantly reduced the sensitivity of mGluR1α not only to [Ca²⁺]_o and [Gd³⁺]_o but also, in some cases, to Glu. The dual activation of mGluR1α by [Ca²⁺]_o and Glu has important implications for the activation of other mGluR subtypes and related receptors. It also opens up new avenues for developing allosteric modulators of mGluR function that target specific human diseases.     

The roles of Ca2+, downstream protein kinases, and oscillatory signaling in regulating fertilization and the activation of development

Reviews in Developmental Biology have covered the pathways that generate the all-important intracellular calcium (Ca²⁺) signal at fertilization [Miyazaki, S., Shirakawa, H., Nakada, K., Honda, Y., 1993a. Essential role of the inositol 1,4,5-trisphosphate receptor/Ca²⁺ release channel in Ca²⁺ waves and Ca²⁺ oscillations at fertilization of mammalian eggs. Dev. Biol. 158, 62-78; Runft, L., Jaffe, L., Mehlmann, L., 2002. Egg activation at fertilization: where it all begins. Dev. Biol. 245, 237-254] and the different temporal responses of Ca²⁺ in many organisms [Stricker, S., 1999. Comparative biology of calcium signaling during fertilization and egg activation in animals. Dev. Biol. 211, 157-176]. Those reviews raise the importance of identifying how Ca²⁺ causes the events of egg activation (EEA) and to what extent these temporal Ca²⁺ responses encode developmental information. This review covers recent studies that have analyzed how these Ca²⁺ signals are interpreted by specific proteins, and how these proteins regulate various EEA responsible for the onset of development. Many of these proteins are protein kinases (CaMKII, PKC, MPF, MAPK, MLCK) whose activity is directly or indirectly regulated by Ca²⁺, and whose amount increases during late oocyte maturation. We cover biochemical progress in defining the signaling pathways between Ca²⁺ and the EEA, as well as discuss how oscillatory or multiple Ca²⁺ signals are likely to have specific advantages biochemically and/or developmentally. These emerging concepts are put into historical context, emphasizing that key contributions have come from many organisms. The intricate interdependence of Ca²⁺, Ca²⁺-dependent proteins, and the EEA raise many new questions for future investigations that will provide insight into the extent to which fertilization-associated signaling has long-range implications for development. In addition, answers to these questions should be beneficial to establishing parameters of egg quality for human and animal IVF, as well as improving egg activation protocols for somatic cell nuclear transfer to generate stem cells and save endangered species.     

An Essential Role for the K+-dependent Na+/Ca2+-exchanger,NCKX4, in Melanocortin-4-receptor-dependent Satiety

K⁺-dependent Na⁺/Ca²⁺-exchangers are broadly expressed in various tissues, and particularly enriched in neurons of the brain. The distinct physiological roles for the different members of this Ca²⁺ transporter family are, however, not well described. Here we show that gene-targeted mice lacking the K⁺-dependent Na⁺/Ca²⁺-exchanger, NCKX4 (gene slc24a4 or Nckx4), display a remarkable anorexia with severe hypophagia and weight loss. Feeding and satiety are coordinated centrally by melanocortin-4 receptors (MC4R) in neurons of the hypothalamic paraventricular nucleus (PVN). The hypophagic response of Nckx4 knock-out mice is accompanied by hyperactivation of neurons in the PVN, evidenced by high levels of c-Fos expression. The activation of PVN neurons in both fasted Nckx4 knock-out and glucose-injected wild-type animals is blocked by Ca²⁺ removal and MC4R antagonists. In cultured hypothalamic neurons, melanocyte stimulating hormone induces an MC4R-dependent and sustained Ca²⁺ signal, which requires phospholipase C activity and plasma membrane Ca²⁺ entry. The Ca²⁺ signal is enhanced in hypothalamic neurons from Nckx4 knock-out animals, and is depressed in cells in which NCKX4 is overexpressed. Finally, MC4R-dependent oxytocin expression in the PVN, a key essential step in satiety, is prevented by blocking phospholipase C activation or Ca²⁺ entry. These findings highlight an essential, and to our knowledge previously unknown, role for Ca²⁺ signaling in the MC4R pathway that leads to satiety, and a novel non-redundant role for NCKX4-mediated Ca²⁺ extrusion in controlling MC4R signaling and feeding behavior. Together, these findings highlight a novel pathway that potentially could be exploited to develop much needed new therapeutics to tackle eating disorders and obesity.     

Store-operated Ca2+ Entry in Malignant Hyperthermia-susceptible Human Skeletal Muscle

In malignant hyperthermia (MH), mutations in RyR1 underlie direct activation of the channel by volatile anesthetics, leading to muscle contracture and a life-threatening increase in core body temperature. The aim of the present study was to establish whether the associated depletion of sarcoplasmic reticulum (SR) Ca²⁺ triggers sarcolemmal Ca²⁺ influx via store-operated Ca²⁺ entry (SOCE). Samples of vastus medialis muscle were obtained from patients undergoing assessment for MH susceptibility using the in vitro contracture test. Single fibers were mechanically skinned, and confocal microscopy was used to detect changes in [Ca²⁺] either within the resealed t-system ([Ca²⁺]_t-sys) or within the cytosol. In normal fibers, halothane (0.5 mm) failed to initiate SR Ca²⁺ release or Ca²⁺_t-sys depletion. However, in MH-susceptible (MHS) fibers, halothane induced both SR Ca²⁺ release and Ca²⁺_t-sys depletion, consistent with SOCE. In some MHS fibers, halothane-induced SR Ca²⁺ release took the form of a propagated wave, which was temporally coupled to a wave of Ca²⁺_t-sys depletion. SOCE was potently inhibited by “extracellular” application of a STIM1 antibody trapped within the t-system but not when the antibody was denatured by heating. In conclusion, (i) in human MHS muscle, SR Ca²⁺ depletion induced by a level of volatile anesthetic within the clinical range is sufficient to induce SOCE, which is tightly coupled to SR Ca²⁺ release; (ii) sarcolemmal STIM1 has an important role in regulating SOCE; and (iii) sustained SOCE from an effectively infinite extracellular Ca²⁺ pool may contribute to the maintained rise in cytosolic [Ca²⁺] that underlies MH.     

TRPC3 Regulates Agonist-stimulated Ca2+ Mobilization by Mediating the Interaction between Type I Inositol 1,4,5-Trisphosphate Receptor, RACK1, and Orai1

There is a body of evidence suggesting that Ca²⁺ handling proteins assemble into signaling complexes required for a fine regulation of Ca²⁺ signals, events that regulate a variety of critical cellular processes. Canonical transient receptor potential (TRPC) and Orai proteins have both been proposed to form Ca²⁺-permeable channels mediating Ca²⁺ entry upon agonist stimulation. A number of studies have demonstrated that inositol 1,4,5-trisphosphate receptors (IP₃Rs) interact with plasma membrane TRPC channels; however, at present there is no evidence supporting the interaction between Orai proteins and IP₃Rs. Here we report that treatment with thapsigargin or cellular agonists results in association of Orai1 with types I and II IP₃Rs. In addition, we have found that TRPC3, RACK1 (receptor for activated protein kinase C-1), and STIM1 (stromal interaction molecule 1) interact with Orai1 upon stimulation with agonists. TRPC3 expression silencing prevented both the interaction of Orai1 with TRPC3 and, more interestingly, the association of Orai1 with the type I IP₃R, but not with the type II IP₃R, thus suggesting that TRPC3 selectively mediates interaction between Orai1 and type I IP₃R. In addition, TRPC3 expression silencing attenuated ATP- and CCh-stimulated interaction between RACK1 and the type I IP₃R, as well as Ca²⁺ release and entry. In conclusion, our results indicate that agonist stimulation results in the formation of an Orai1-STIM1-TRPC3-RACK1-type I IP₃R complex, where TRPC3 plays a central role. This Ca²⁺ signaling complex might be important for both agonist-induced Ca²⁺ release and entry.     

Protein Kinase C�� Negatively Regulates Store-independent Ca2+ Entry and Phosphatidylserine Exposure Downstream of Glycoprotein VI in Platelets

Platelet activation must be tightly controlled to provide an effective, but not excessive, response to vascular injury. Cytosolic calcium is a critical regulator of platelet function, including granule secretion, integrin activation, and phosphatidylserine (PS) exposure. Here we report that the novel protein kinase C isoform, PKCθ, plays an important role in negatively regulating Ca²⁺ signaling downstream of the major collagen receptor, glycoprotein VI (GPVI). This limits PS exposure and so may prevent excessive platelet procoagulant activity. Stimulation of GPVI resulted in significantly higher and more sustained Ca²⁺ signals in PKCθ^−/− platelets. PKCθ acts at multiple distinct sites. PKCθ limits secretion, reducing autocrine ADP signaling that enhances Ca²⁺ release from intracellular Ca²⁺ stores. PKCθ thereby indirectly regulates activation of store-operated Ca²⁺ entry. However, PKCθ also directly and negatively regulates store-independent Ca²⁺ entry. This pathway, activated by the diacylglycerol analogue, 1-oleoyl-2-acetyl-sn-glycerol, was enhanced in PKCθ^−/− platelets, independently of ADP secretion. Moreover, LOE-908, which blocks 1-oleoyl-2-acetyl-sn-glycerol-induced Ca²⁺ entry but not store-operated Ca²⁺ entry, blocked the enhanced GPVI-dependent Ca²⁺ signaling and PS exposure seen in PKCθ^−/− platelets. We propose that PKCθ normally acts to restrict store-independent Ca²⁺ entry during GPVI signaling, which results in reduced PS exposure, limiting platelet procoagulant activity during thrombus formation.     

Polyspermy block in jellyfish eggs: Collaborative controls by Ca and MAPK

Jellyfish eggs neither undergo apparent cortical reaction nor show any significant change in the membrane potential at fertilization, but nevertheless show monospermy. Utilizing the perfectly transparent eggs of the hydrozoan jellyfish Cytaeis uchidae, here we show that the polyspermy block is accomplished via a novel mechanism: a collaboration between Ca²⁺ and mitogen-activated protein kinase (MAPK). In Cytaeis, adhesion of a sperm to the animal pole surface of an egg was immediately followed by sperm–egg fusion and initiation of an intracellular Ca²⁺ rise from this site. The elevated Ca²⁺ levels lasted for several minutes following the sperm–egg fusion. The Ca²⁺ rise proved to be necessary and sufficient for a polyspermy block, as inhibiting a Ca²⁺ rise with EGTA promoted polyspermy, and conversely, triggering a Ca²⁺ rise by inositol 1,4,5-trisphosphate (IP₃) or excess K⁺ immediately abolished the egg’s capacity for sperm–egg fusion. A Ca²⁺ rise at fertilization or by artificial stimulations evoked dephosphorylation of MAPK in eggs. The eggs in which phosphorylated MAPK was maintained by injection of mRNA for MAPK kinase kinase (Mos), like intact eggs, exhibited a Ca²⁺ rise at fertilization or by IP₃ injection, and shut down the subsequent sperm–egg fusion. However, the Mos-expressing eggs became capable of accepting sperm following the arrest of Ca²⁺ rise. In contrast, addition of inhibitors of MAPK kinase (MEK) to unfertilized eggs caused MAPK dephosphorylation without elevating Ca²⁺ levels, and prevented sperm–egg fusion. Rephosphorylation of MAPK by injecting Mos mRNA after fertilization recovered sperm attraction, which is known to be another MAPK-dependent event, but did not permit subsequent sperm–egg fusion. Thus, it is possible that MAPK dephosphorylation irreversibly blocks sperm–egg fusion and reversibly suppresses sperm attraction. Collectively, our data suggest that both the fast and late mechanisms dependent on Ca²⁺ and MAPK, respectively, ensure a polyspermy block in jellyfish eggs.