Transient Receptor Potential Melastatin 7 (TRPM7) Ion Channel Inhibitors: Preliminary SAR and Conformational Studies of Xenicane Diterpenoids from the Hawaiian Soft Coral Sarcothelia edmondsoni.

Waixenicin A, a xenicane diterpene from the octocoral Sarcothelia edmondsoni, is a selective, potent inhibitor of the TRPM7 ion channel. To study the structure-activity relationship (SAR) of waixenicin A, we isolated and assayed related diterpenes from S. edmondsoni. In addition to known waixenicins A (1) and B (2), we purified six xenicane diterpenes, 7S,8S-epoxywaixenicins A (3) and B (4), 12-deacetylwaixenicin A (5), waixenicin E (6), waixenicin F (7), and 20-acetoxyxeniafaraunol B (8). We elucidated the structures of 3-8 by NMR and MS analyses. Compounds 1, 2, 3, 4, and 6 inhibited TRPM7 activity in a cell-based assay, while 5, 7, and 8 were inactive. A preliminary SAR emerged showing that alterations to the nine-membered ring of 1 did not reduce activity, while the 12-acetoxy group, in combination with the dihydropyran, appears to be necessary for TRPM7 inhibition. The bioactive compounds are proposed to be latent electrophiles by formation of a conjugated oxocarbenium ion intermediate. Whole-cell patch-clamp experiments demonstrated that waixenicin A inhibition is irreversible, consistent with a covalent inhibitor, and showed nanomolar potency for waixenicin B (2). Conformational analysis (DFT) of 1, 3, 7, and 8 revealed insights into the conformation of waixenicin A and congeners and provided information regarding the stabilization of the proposed pharmacophore.

The TRPM7 kinase limits receptor-induced calcium release by regulating heterotrimeric G-proteins.

The melastatin-related transient receptor potential member 7 (TRPM7) is a unique fusion protein with both ion channel function and enzymatic α-kinase activity. TRPM7 is essential for cellular systemic magnesium homeostasis and early embryogenesis; it promotes calcium transport during global brain ischemia and emerges as a key player in cancer growth. TRPM7 channels are negatively regulated through G-protein-coupled receptor-stimulation, either by reducing cellular cyclic adenosine monophosphate (cAMP) or depleting phosphatidylinositol bisphosphate (PIP2) levels in the plasma membrane. We here identify that heterologous overexpression of human TRPM7-K1648R mutant will lead to disruption of protease or purinergic receptor-induced calcium release. The disruption occurs at the level of Gq, which requires intact TRPM7 kinase phosphorylation activity for orderly downstream signal transduction to activate phospholipase (PLC)ß and cause calcium release. We propose that this mechanism may support limiting GPCR-mediated calcium signaling in times of insufficient cellular ATP supply.

The TRPM7 channel kinase regulates store-operated calcium entry.

KEY POINTS: Pharmacological and molecular inhibition of transient receptor potential melastatin 7 (TRPM7) reduces store-operated calcium entry (SOCE). Overexpression of TRPM7 in TRPM7-/- cells restores SOCE. TRPM7 is not a store-operated calcium channel. TRPM7 kinase rather than channel modulates SOCE. TRPM7 channel activity contributes to the maintenance of store Ca2+ levels at rest. ABSTRACT: The transient receptor potential melastatin 7 (TRPM7) is a protein that combines an ion channel with an intrinsic kinase domain, enabling it to modulate cellular functions either by conducting ions through the pore or by phosphorylating downstream proteins via its kinase domain. In the present study, we report store-operated calcium entry (SOCE) as a novel target of TRPM7 kinase activity. TRPM7-deficient chicken DT40 B lymphocytes exhibit a strongly impaired SOCE compared to wild-type cells as a result of reduced calcium release activated calcium currents, and independently of potassium channel regulation, membrane potential changes or changes in cell-cycle distribution. Pharmacological blockade of TRPM7 with NS8593 or waixenicin A in wild-type B lymphocytes results in a significant decrease in SOCE, confirming that TRPM7 activity is acutely linked to SOCE, without TRPM7 representing a store-operated channel itself. Using kinase-deficient mutants, we find that TRPM7 regulates SOCE through its kinase domain. Furthermore, Ca2+ influx through TRPM7 is essential for the maintenance of endoplasmic reticulum Ca2+ concentration in resting cells, and for the refilling of Ca2+ stores after a Ca2+ signalling event. We conclude that the channel kinase TRPM7 and SOCE are synergistic mechanisms regulating intracellular Ca2+ homeostasis.

Inhibition of TRPM7 suppresses cell proliferation of colon adenocarcinoma in vitro and induces hypomagnesemia in vivo without affecting azoxymethane-induced early colon cancer in mice.

BACKGROUND: Magnesium (Mg2+) is an essential cation implicated in carcinogenesis, solid tumor progression and metastatic potential. The Transient Receptor Potential Melastatin Member 7 (TRPM7) is a divalent ion channel involved in cellular and systemic Mg2+ homeostasis. Abnormal expression of TRPM7 is found in numerous cancers, including colon, implicating TRPM7 in this process. METHODS: To establish a possible link between systemic magnesium (Mg2+) status, the Mg2+ conducting channel TRPM7 in colon epithelial cells, and colon carcinogenesis, in vitro whole-cell patch clamp electrophysiology, qPCR, and pharmacological tools were used probing human colorectal adenocarcinoma HT-29 as well as normal primary mouse colon epithelial cells. This was extended to and combined with aberrant crypt foci development in an azoxymethane-induced colorectal cancer mouse model under hypomagnesemia induced by diet or pharmacologic intervention. RESULTS: We find that TRPM7 drives colon cancer cell proliferation in human HT-29 and expresses in normal primary mouse colon epithelia. This is linked to TRPM7's dominant role as Mg2+ transporter, since high extracellular Mg2+ supplementation cannot rescue inhibition of cell proliferation caused by suppressing TRPM7 either genetically or pharmacologically. In vivo experiments in mice provide evidence that the specific TRPM7 inhibitor waixenicin A, given as a single bolus injection, induces transient hypomagnesemia and increases intestinal absorption of calcium. Repeated injections of waixenicin A over 3 weeks cause hypomagnesemia via insufficient Mg2+ absorption by the colon. However, neither waixenicin A, nor a diet low in Mg2+, affect aberrant crypt foci development in an azoxymethane-induced colorectal cancer mouse model. CONCLUSION: Early stage colon cancer proceeds independent of systemic Mg2+ status and TRPM7, and waixenicin A is a useful pharmacological tool to study of TRPM7 in vitro and in vivo.

Scalaradial Is a Potent Inhibitor of Transient Receptor Potential Melastatin 2 (TRPM2) Ion Channels.

TRPM2 is a Ca2+-permeable, nonselective cation channel that plays a role in oxidant-induced cell death, insulin secretion, and cytokine release. Few TRPM2 inhibitors have been reported, which hampers the validation of TRPM2 as a drug target. While screening our in-house marine-derived chemical library, we identified scalaradial and 12-deacetylscalaradial as the active components within an extract of an undescribed species of Cacospongia (class Demospongiae, family Thorectidae) that strongly inhibited TRPM2-mediated Ca2+ influx in TRPM2-overexpressing HEK293 cells. In whole-cell patch-clamp experiments, scalaradial (and similarly 12-deacetylscalaradial) inhibited TRPM2-mediated currents in a concentration- and time-dependent manner (∼20 min to full onset; IC50 210 nM). Scalaradial inhibited TRPM7 with less potency (IC50 760 nM) but failed to inhibit CRAC, TRPM4, and TRPV1 currents in whole-cell patch clamp experiments. Scalaradial's effect on TRPM2 channels was shown to be independent of its well-known ability to inhibit secreted phospholipase A2 (sPLA2) and its reported effects on extracellular signal-regulated kinases (ERK) and Akt pathways. In addition, scalaradial was shown to inhibit endogenous TRPM2 currents in a rat insulinoma cell line (IC50 330 nM). Based on its potency and emerging specificity profile, scalaradial is an important addition to the small number of known TRPM2 inhibitors.

TRPM7 kinase activity regulates murine mast cell degranulation.

KEY POINTS: The Mg(2+) and Ca(2+) conducting transient receptor potential melastatin 7 (TRPM7) channel-enzyme (chanzyme) has been implicated in immune cell function. Mice heterozygous for a TRPM7 kinase deletion are hyperallergic, while mice with a single point mutation at amino acid 1648, silencing kinase activity, are not. As mast cell mediators trigger allergic reactions, we here determine the function of TRPM7 in mast cell degranulation and histamine release. Our data establish that TRPM7 kinase activity regulates mast cell degranulation and release of histamine independently of TRPM7 channel function. Our findings suggest a regulatory role of TRPM7 kinase activity on intracellular Ca(2+) and extracellular Mg(2+) sensitivity of mast cell degranulation. ABSTRACT: Transient receptor potential melastatin 7 (TRPM7) is a divalent ion channel with a C-terminally located α-kinase. Mice heterozygous for a TRPM7 kinase deletion (TRPM7(+/∆K) ) are hypomagnesaemic and hyperallergic. In contrast, mice carrying a single point mutation at amino acid 1648, which silences TRPM7 kinase activity (TRPM7(KR) ), are not hyperallergic and are resistant to systemic magnesium (Mg(2+) ) deprivation. Since allergic reactions are triggered by mast cell-mediated histamine release, we investigated the function of TRPM7 on mast cell degranulation and histamine release using wild-type (TRPM7(+/+) ), TRPM7(+/∆K) and TRPM7(KR) mice. We found that degranulation and histamine release proceeded independently of TRPM7 channel function. Furthermore, extracellular Mg(2+) assured unperturbed IgE-DNP-dependent exocytosis, independently of TRPM7. However, impairment of TRPM7 kinase function suppressed IgE-DNP-dependent exocytosis, slowed the cellular degranulation rate, and diminished the sensitivity to intracellular calcium (Ca(2+) ) in G protein-induced exocytosis. In addition, G protein-coupled receptor (GPCR) stimulation revealed strong suppression of histamine release, whereas removal of extracellular Mg(2+) caused the phenotype to revert. We conclude that the TRPM7 kinase activity regulates murine mast cell degranulation by changing its sensitivity to intracellular Ca(2+) and affecting granular mobility and/or histamine contents.

Human CNNM2 is not a Mg(2+) transporter per se.

CNNM2 is associated with the regulation of serum Mg concentration, and when mutated, with severe familial hypomagnesemia. The function and cellular localization of CNNM2 and its isomorphs (Iso) remain controversial. The objective of this work was to examine the following: (1) the transcription-responsiveness of CNNM2 to Mg starvation, (2) the cellular localization of Iso1 and Iso2, (3) the ability of Iso1 and Iso2 to transport Mg(2+), and (4) the complex-forming ability and spectra of potential interactors of Iso1 and Iso2. The five main findings are as follows. (1) Mg-starvation induces CNNM2 overexpression that is markedly higher in JVM-13 cells (lymphoblasts) compared with Jurkat cells (T-lymphocytes). (2) Iso1 and Iso2 localize throughout various subcellular compartments in transgenic HEK293 cells overexpressing Iso1 or Iso2. (3) Iso1 and Iso2 do not transport Mg(2+) in an electrogenic or electroneutral mode in transgenic HEK293 cells overexpressing Iso1 or Iso2. (4) Both Iso1 and Iso2 form complexes of a higher molecular order. (5) The spectrum of potential interactors of Iso1 is ten times smaller than that of Iso2. We conclude that sensitivity of CNNM2 expression to extracellular Mg(2+) depletion depends on cell type. Iso1 and Iso2 exhibit a dispersed pattern of cellular distribution; thus, they are not exclusively integral to the cytoplasmic membrane. Iso1 and Iso2 are not Mg(2+) transporters per se. Both isomorphs form protein complexes, and divergent spectra of potential interactors of Iso1 and Iso2 indicate that each isomorph has a distinctive function. CNNM2 is therefore the first ever identified Mg(2+) homeostatic factor without being a Mg(2+) transporter per se.

The TRPM6 kinase domain determines the Mg·ATP sensitivity of TRPM7/M6 heteromeric ion channels.

The transient receptor potential melastatin member 7 (TRPM7) and member 6 (TRPM6) are divalent cation channel kinases essential for magnesium (Mg(2+)) homeostasis in vertebrates. It remains unclear how TRPM6 affects divalent cation transport and whether this involves functional homomeric TRPM6 plasma membrane channels or heteromeric channel assemblies with TRPM7. We show that homomeric TRPM6 is highly sensitive to intracellular free Mg(2+) and therefore unlikely to be active at physiological levels of [Mg(2+)]i. Co-expression of TRPM7 and TRPM6 produces heteromeric TRPM7/M6 channels with altered pharmacology and sensitivity to intracellular Mg·ATP compared with homomeric TRPM7. Strikingly, the activity of heteromeric TRPM7/M6 channels is independent of intracellular Mg·ATP concentrations, essentially uncoupling channel activity from cellular energy status. Disruption of TRPM6 kinase phosphorylation activity re-introduces Mg·ATP sensitivity to the heteromeric channel similar to that of TRPM7. Thus, TRPM6 modulates the functionality of TRPM7, and the TRPM6 kinase plays a critical role in tuning the phenotype of the TRPM7·M6 channel complex.

ORAI3 silencing alters cell proliferation and cell cycle progression via c-myc pathway in breast cancer cells.

Members of the Orai family are highly selective calcium ion channels that play an important role in store-operated calcium entry. Among the three known Orai isoforms, Orai3 has gained increased attention, notably for its emerging role in cancer. We recently demonstrated that Orai3 channels are over-expressed in breast cancer (BC) biopsies, and involved specifically in proliferation, cell cycle progression and survival of MCF-7 BC cells. Here, we investigate the downstream signaling mechanisms affected by Orai3 silencing, leading to the subsequent functional impact specifically seen in MCF-7 cancer cells. We report a correlation between Orai3 and c-myc expression in tumor tissues and in the MCF-7 cancer cell line by demonstrating that Orai3 down-regulation reduces both expression and activity of the proto-oncogene c-myc. This is likely mediated through the MAP Kinase pathway, as we observed decreased pERK1/2 levels and cell-cycle arrest in G1 phase after Orai3 silencing. Our results provide strong evidence that the c-myc proto-oncogene is influenced by the store-operated calcium entry channel Orai3 through the MAP kinase pathway. This connection provides new clues in the downstream mechanism linking Orai3 channels and proliferation, cell cycle progression and survival of MCF-7 BC cells.

TRPM7 is regulated by halides through its kinase domain.

Transient receptor potential melastatin 7 (TRPM7) is a divalent-selective cation channel fused to an atypical α-kinase. TRPM7 is a key regulator of cell growth and proliferation, processes accompanied by mandatory cell volume changes. Osmolarity-induced cell volume alterations regulate TRPM7 through molecular crowding of solutes that affect channel activity, including magnesium (Mg(2+)), Mg-nucleotides and a further unidentified factor. Here, we assess whether chloride and related halides can act as negative feedback regulators of TRPM7. We find that chloride and bromide inhibit heterologously expressed TRPM7 in synergy with intracellular Mg(2+) ([Mg(2+)]i) and this is facilitated through the ATP-binding site of the channel's kinase domain. The synergistic block of TRPM7 by chloride and Mg(2+) is not reversed during divalent-free or acidic conditions, indicating a change in protein conformation that leads to channel inactivation. Iodide has the strongest inhibitory effect on TRPM7 at physiological [Mg(2+)]i. Iodide also inhibits endogenous TRPM7-like currents as assessed in MCF-7 breast cancer cells, where upregulation of SLC5A5 sodium-iodide symporter enhances iodide uptake and inhibits cell proliferation. These results indicate that chloride could be an important factor in modulating TRPM7 during osmotic stress and implicate TRPM7 as a possible molecular mechanism contributing to the anti-proliferative characteristics of intracellular iodide accumulation in cancer cells.

TRPM2.

TRPM2 is the second member of the transient receptor potential melastatin-related (TRPM) family of cation channels. The protein is widely expressed including in the brain, immune system, endocrine cells, and endothelia. It embodies both ion channel functionality and enzymatic ADP-ribose (ADPr) hydrolase activity. TRPM2 is a Ca(2+)-permeable nonselective cation channel embedded in the plasma membrane and/or lysosomal compartments that is primarily activated in a synergistic fashion by intracellular ADP-ribose (ADPr) and Ca(2+). It is also activated by reactive oxygen and nitrogen species (ROS/NOS) and enhanced by additional factors, such as cyclic ADPr and NAADP, while inhibited by permeating protons (acidic pH) and adenosine monophosphate (AMP). Activation of TRPM2 leads to increases in intracellular Ca(2+) levels, which can serve signaling roles in inflammatory and secretory cells through release of vesicular mediators (e.g., cytokines, neurotransmitters, insulin) and in extreme cases can induce apoptotic and necrotic cell death under oxidative stress.

Cannabigerolic Acid (CBGA) Inhibits the TRPM7 Ion Channel Through its Kinase Domain.

Cannabinoids are a major class of compounds produced by the plant Cannabis sativa. Previous work has demonstrated that the main cannabinoids cannabidiol (CBD) and tetrahydrocannabinol (THC) can have some beneficial effects on pain, inflammation, epilepsy, and chemotherapy-induced nausea and vomiting. While CBD and THC represent the two major plant cannabinoids, some hemp varieties with enzymatic deficiencies produce mainly cannabigerolic acid (CBGA). We recently reported that CBGA has a potent inhibitory effect on both Store-Operated Calcium Entry (SOCE) via inhibition of Calcium Release-Activated Calcium (CRAC) channels as well as currents carried by the channel-kinase TRPM7. Importantly, CBGA prevented kidney damage and suppressed mRNA expression of inflammatory cytokines through inhibition of these mechanisms in an acute nephropathic mouse model. In the present study, we investigate the most common major and minor cannabinoids to determine their potential efficacy on TRPM7 channel function. We find that approximately half of the tested cannabinoids suppress TRPM7 currents to some degree, with CBGA having the strongest inhibitory effect on TRPM7. We determined that the CBGA-mediated inhibition of TRPM7 requires a functional kinase domain, is sensitized by both intracellular Mg⋅ATP and free Mg2+ and reduced by increases in intracellular Ca2+. Finally, we demonstrate that CBGA inhibits native TRPM7 channels in a B lymphocyte cell line. In conclusion, we demonstrate that CBGA is the most potent cannabinoid in suppressing TRPM7 activity and possesses therapeutic potential for diseases in which TRPM7 is known to play an important role such as cancer, stroke, and kidney disease.

STIM2 drives Ca2+ oscillations through store-operated Ca2+ entry caused by mild store depletion.

Abstract Agonist-induced Ca(2+) oscillations in many cell types are triggered by Ca(2+) release from intracellular stores and driven by store-operated Ca(2+) entry. Stromal cell-interaction molecule (STIM) 1 and STIM2 serve as endoplasmic reticulum Ca(2+) sensors that, upon store depletion, activate Ca(2+) release-activated Ca(2+) channels (Orai1-3, CRACM1-3) in the plasma membrane. However, their relative roles in agonist-mediated Ca(2+) oscillations remain ambiguous. Here we report that while both STIM1 and STIM2 contribute to store-refilling during Ca(2+) oscillations in mast cells (RBL), T cells (Jurkat) and human embryonic kidney (HEK293) cells, they do so dependent on the level of store depletion. Molecular silencing of STIM2 by siRNA or inhibition by G418 suppresses store-operated Ca(2+) entry and agonist-mediated Ca(2+) oscillations at low levels of store depletion, without interfering with STIM1-mediated signals induced by full store depletion. Thus, STIM2 is preferentially activated by low-level physiological agonist concentrations that cause mild reductions in endoplasmic reticulum Ca(2+) levels. We conclude that with increasing agonist concentrations, store-operated Ca(2+) entry is mediated initially by endogenous STIM2 and incrementally by STIM1, enabling differential modulation of Ca(2+) entry over a range of agonist concentrations and levels of store depletion.

Amplification of CRAC current by STIM1 and CRACM1 (Orai1).

Depletion of intracellular calcium stores activates store-operated calcium entry across the plasma membrane in many cells. STIM1, the putative calcium sensor in the endoplasmic reticulum, and the calcium release-activated calcium (CRAC) modulator CRACM1 (also known as Orai1) in the plasma membrane have recently been shown to be essential for controlling the store-operated CRAC current (I(CRAC)). However, individual overexpression of either protein fails to significantly amplify I(CRAC). Here, we show that STIM1 and CRACM1 interact functionally. Overexpression of both proteins greatly potentiates I(CRAC), suggesting that STIM1 and CRACM1 mutually limit store-operated currents and that CRACM1 may be the long-sought CRAC channel.

CBGA ameliorates inflammation and fibrosis in nephropathy.

Cannabidiol (CBD) is thought to have multiple biological effects, including the ability to attenuate inflammatory processes. Cannabigerols (CBGA and its decarboxylated CBG molecule) have pharmacological profiles similar to CBD. The endocannabinoid system has recently emerged to contribute to kidney disease, however, the therapeutic properties of cannabinoids in kidney disease remain largely unknown. In this study, we determined whether CBD and CBGA can attenuate kidney damage in an acute kidney disease model induced by the chemotherapeutic cisplatin. In addition, we evaluated the anti-fibrosis effects of these cannabinoids in a chronic kidney disease model induced by unilateral ureteral obstruction (UUO). We find that CBGA, but not CBD, protects the kidney from cisplatin-induced nephrotoxicity. CBGA also strongly suppressed mRNA of inflammatory cytokines in cisplatin-induced nephropathy, whereas CBD treatment was only partially effective. Furthermore, both CBGA and CBD treatment significantly reduced apoptosis through inhibition of caspase-3 activity. In UUO kidneys, both CBGA and CBD strongly reduced renal fibrosis. Finally, we find that CBGA, but not CBD, has a potent inhibitory effect on the channel-kinase TRPM7. We conclude that CBGA and CBD possess reno-protective properties, with CBGA having a higher efficacy, likely due to its dual anti-inflammatory and anti-fibrotic effects paired with TRPM7 inhibition.

Waixenicin A inhibits cell proliferation through magnesium-dependent block of transient receptor potential melastatin 7 (TRPM7) channels.

Transient receptor potential melastatin 7 (TRPM7) channels represent the major magnesium-uptake mechanism in mammalian cells and are key regulators of cell growth and proliferation. They are expressed abundantly in a variety of human carcinoma cells controlling survival, growth, and migration. These characteristics are the basis for recent interest in the channel as a target for cancer therapeutics. We screened a chemical library of marine organism-derived extracts and identified waixenicin A from the soft coral Sarcothelia edmondsoni as a strong inhibitor of overexpressed and native TRPM7. Waixenicin A activity was cytosolic and potentiated by intracellular free magnesium (Mg(2+)) concentration. Mutating a Mg(2+) binding site on the TRPM7 kinase domain reduced the potency of the compound, whereas kinase deletion enhanced its efficacy independent of Mg(2+). Waixenicin A failed to inhibit the closely homologous TRPM6 channel and did not significantly affect TRPM2, TRPM4, and Ca(2+) release-activated Ca(2+) current channels. Therefore, waixenicin A represents the first potent and relatively specific inhibitor of TRPM7 ion channels. Consistent with TRPM7 inhibition, the compound blocked cell proliferation in human Jurkat T-cells and rat basophilic leukemia cells. Based on the ability of the compound to inhibit cell proliferation through Mg(2+)-dependent block of TRPM7, waixenicin A, or structural analogs may have cancer-specific therapeutic potential, particularly because certain cancers accumulate cytosolic Mg(2+).

Dendritic cell maturation and chemotaxis is regulated by TRPM2-mediated lysosomal Ca2+ release.

Chemokines induce calcium (Ca(2+)) signaling and chemotaxis in dendritic cells (DCs), but the molecular players involved in shaping intracellular Ca(2+) changes remain to be characterized. Using siRNA and knockout mice, we show that in addition to inositol 1,4,5-trisphosphate (IP(3))-mediated Ca(2+) release and store-operated Ca(2+) entry (SOCE), the transient receptor potential melastatin 2 (TRPM2) channel contributes to Ca(2+) release but not Ca(2+) influx in mouse DCs. Consistent with these findings, TRPM2 expression in DCs is restricted to endolysosomal vesicles, whereas in neutrophils, the channel localizes to the plasma membrane. TRPM2-deficient DCs show impaired maturation and severely compromised chemokine-activated directional migration as well as bacterial-induced DC trafficking to the draining lymph nodes. Defective DC chemotaxis is due to perturbed chemokine-receptor-initiated Ca(2+) signaling mechanisms, which include suppression of TRPM2-mediated Ca(2+) release and secondary modification of SOCE. DCs deficient in both TRPM2 and IP(3) receptor signaling lose their ability to perform chemotaxis entirely. These results highlight TRPM2 as a key player regulating DC chemotaxis through its function as Ca(2+) release channel and confirm ADP-ribose as a novel second messenger for intracellular Ca(2+) mobilization.

Stimulation of Ca2+-channel Orai1/STIM1 by serum- and glucocorticoid-inducible kinase 1 (SGK1).

Ca(2+) signaling includes store-operated Ca(2+) entry (SOCE) following depletion of endoplasmic reticulum (ER) Ca(2+) stores. On store depletion, the ER Ca(2+) sensor STIM1 activates Orai1, the pore-forming unit of Ca(2+)-release-activated Ca(2+) (CRAC) channels. Here, we show that Orai1 is regulated by serum- and glucocorticoid-inducible kinase 1 (SGK1), a growth factor-regulated kinase. Membrane Orai1 protein abundance, I(CRAC), and SOCE in human embryonic kidney (HEK293) cells stably expressing Orai1 and transfected with STIM1 were each significantly enhanced by coexpression of constitutively active (S422D)SGK1 (by+81, +378, and+136%, respectively) but not by inactive (K127N)SGK1. Coexpression of the ubiquitin ligase Nedd4-2, an established negatively regulated SGK1 target, down-regulated SOCE (by -48%) and I(CRAC) (by -60%), an effect reversed by expression of (S422D)SGK1 (by +175 and +173%, respectively). Orai1 protein abundance and SOCE were significantly lower in mast cells from SGK1-knockout (sgk1(-/-)) mice (by -37% and -52%, respectively) than in mast cells from wild-type (sgk1(+/+)) littermates. Activation of SOCE by sarcoplasmic/endoplasmic reticulum Ca(2+)-ATPase-inhibitor thapsigargin (2 µM) stimulated migration, an effect significantly higher (by +306%) in (S422D)SGK1-expressing than in (K127N)SGK1-expressing HEK293 cells, and also significantly higher (by +108%) in sgk1(+/+) than in sgk1(-/-) mast cells. SGK1 is thus a novel key player in the regulation of SOCE.

Acidic Cannabinoids Suppress Proinflammatory Cytokine Release by Blocking Store-operated Calcium Entry.

Cannabis sativa has long been known to affect numerous biological activities. Although plant extracts, purified cannabinoids, or synthetic cannabinoid analogs have shown therapeutic potential in pain, inflammation, seizure disorders, appetite stimulation, muscle spasticity, and treatment of nausea/vomiting, the underlying mechanisms of action remain ill-defined. In this study we provide the first comprehensive overview of the effects of whole-plant Cannabis extracts and various pure cannabinoids on store-operated calcium (Ca2+) entry (SOCE) in several different immune cell lines. Store-operated Ca2+ entry is one of the most significant Ca2+ influx mechanisms in immune cells, and it is critical for the activation of T lymphocytes, leading to the release of proinflammatory cytokines and mediating inflammation and T cell proliferation, key mechanisms for maintaining chronic pain. While the two major cannabinoids cannabidiol and trans-Δ9-tetrahydrocannabinol were largely ineffective in inhibiting SOCE, we report for the first time that several minor cannabinoids, mainly the carboxylic acid derivatives and particularly cannabigerolic acid, demonstrated high potency against SOCE by blocking calcium release-activated calcium currents. Moreover, we show that this inhibition of SOCE resulted in a decrease of nuclear factor of activated T-cells activation and Interleukin 2 production in human T lymphocytes. Taken together, these results indicate that cannabinoid-mediated inhibition of a proinflammatory target such as SOCE may at least partially explain the anti-inflammatory and analgesic effects of Cannabis.