(-)-Englerin A-evoked Cytotoxicity Is Mediated by Na+ Influx and Counteracted by Na+/K+-ATPase.

(-)-Englerin A ((-)-EA) has a rapid and potent cytotoxic effect on several types of cancer cell that is mediated by plasma membrane ion channels containing transient receptor potential canonical 4 (TRPC4) protein. Because these channels are Ca2+-permeable, it was initially thought that the cytotoxicity arose as a consequence of Ca2+ overload. Here we show that this is not the case and that the effect of (-)-EA is mediated by a heteromer of TRPC4 and TRPC1 proteins. Both TRPC4 and TRPC1 were required for (-)-EA cytotoxicity; however, although TRPC4 was necessary for the (-)-EA-evoked Ca2+ elevation, TRPC1 was not. TRPC1 either had no role or was a negative regulator of Ca2+ entry. By contrast, both TRPC4 and TRPC1 were necessary for monovalent cation entry evoked by (-)-EA, and (-)-EA-evoked cell death was dependent upon entry of the monovalent cation Na+ We therefore hypothesized that Na+/K+-ATPase might act protectively by counteracting the Na+ load resulting from sustained Na+ entry. Indeed, inhibition of Na+/K+-ATPase by ouabain potently and strongly increased (-)-EA-evoked cytotoxicity. The data suggest that (-)-EA achieves cancer cell cytotoxicity by inducing sustained Na+ entry through heteromeric TRPC1/TRPC4 channels and that the cytotoxic effect of (-)-EA can be potentiated by Na+/K+-ATPase inhibition.

Picomolar, selective, and subtype-specific small-molecule inhibition of TRPC1/4/5 channels.

The concentration of free cytosolic Ca2+ and the voltage across the plasma membrane are major determinants of cell function. Ca2+-permeable non-selective cationic channels are known to regulate these parameters, but understanding of these channels remains inadequate. Here we focus on transient receptor potential canonical 4 and 5 proteins (TRPC4 and TRPC5), which assemble as homomers or heteromerize with TRPC1 to form Ca2+-permeable non-selective cationic channels in many mammalian cell types. Multiple roles have been suggested, including in epilepsy, innate fear, pain, and cardiac remodeling, but limitations in tools to probe these channels have restricted progress. A key question is whether we can overcome these limitations and develop tools that are high-quality, reliable, easy to use, and readily accessible for all investigators. Here, through chemical synthesis and studies of native and overexpressed channels by Ca2+ and patch-clamp assays, we describe compound 31, a remarkable small-molecule inhibitor of TRPC1/4/5 channels. Its potency ranged from 9 to 1300 pm, depending on the TRPC1/4/5 subtype and activation mechanism. Other channel types investigated were unaffected, including TRPC3, TRPC6, TRPV1, TRPV4, TRPA1, TRPM2, TRPM8, and store-operated Ca2+ entry mediated by Orai1. These findings suggest identification of an important experimental tool compound, which has much higher potency for inhibiting TRPC1/4/5 channels than previously reported agents, impressive specificity, and graded subtype selectivity within the TRPC1/4/5 channel family. The compound should greatly facilitate future studies of these ion channels. We suggest naming this TRPC1/4/5-inhibitory compound Pico145.

Transient receptor potential canonical 4 and 5 proteins as targets in cancer therapeutics.

Novel approaches towards cancer therapy are urgently needed. One approach might be to target ion channels mediating Ca2+ entry because of the critical roles played by Ca2+ in many cell types, including cancer cells. There are several types of these ion channels, but here we address those formed by assembly of transient receptor potential canonical (TRPC) proteins, particularly those which involve two closely related members of the family: TRPC4 and TRPC5. We focus on these proteins because recent studies point to roles in important aspects of cancer: drug resistance, transmission of drug resistance through extracellular vesicles, tumour vascularisation, and evoked cancer cell death by the TRPC4/5 channel activator (-)-englerin A. We conclude that further research is both justified and necessary before these proteins can be considered as strong targets for anti-cancer cell drug discovery programmes. It is nevertheless already apparent that inhibitors of the channels would be unlikely to cause significant adverse effects, but, rather, have other effects which may be beneficial in the context of cancer and chemotherapy, potentially including suppression of innate fear, visceral pain and pathological cardiac remodelling.

(-)-Englerin†A is a potent and selective activator of TRPC4 and TRPC5 calcium channels.

Current therapies for common types of cancer such as renal cell cancer are often ineffective and unspecific, and novel pharmacological targets and approaches are in high demand. Here we show the unexpected possibility for the rapid and selective killing of renal cancer cells through activation of calcium-permeable nonselective transient receptor potential canonical (TRPC) calcium channels by the sesquiterpene (-)-englerin A. This compound was found to be a highly efficient, fast-acting, potent, selective, and direct stimulator of TRPC4 and TRPC5 channels. TRPC4/5 activation through a high-affinity extracellular (-)-englerin A binding site may open up novel opportunities for drug discovery aimed at renal cancer.

PIEZO1 and PECAM1 interact at cell-cell junctions and partner in endothelial force sensing.

Two prominent concepts for the sensing of shear stress by endothelium are the PIEZO1 channel as a mediator of mechanically activated calcium ion entry and the PECAM1 cell adhesion molecule as the apex of a triad with CDH5 and VGFR2. Here, we investigated if there is a relationship. By inserting a non-disruptive tag in native PIEZO1 of mice, we reveal in situ overlap of PIEZO1 with PECAM1. Through reconstitution and high resolution microscopy studies we show that PECAM1 interacts with PIEZO1 and directs it to cell-cell junctions. PECAM1 extracellular N-terminus is critical in this, but a C-terminal intracellular domain linked to shear stress also contributes. CDH5 similarly drives PIEZO1 to junctions but unlike PECAM1 its interaction with PIEZO1 is dynamic, increasing with shear stress. PIEZO1 does not interact with VGFR2. PIEZO1 is required in Ca2+-dependent formation of adherens junctions and associated cytoskeleton, consistent with it conferring force-dependent Ca2+ entry for junctional remodelling. The data suggest a pool of PIEZO1 at cell junctions, the coming together of PIEZO1 and PECAM1 mechanisms and intimate cooperation of PIEZO1 and adhesion molecules in tailoring junctional structure to mechanical requirement.

Rab46 integrates Ca2+ and histamine signaling to regulate selective cargo release from Weibel-Palade bodies.

Endothelial cells selectively release cargo stored in Weibel-Palade bodies (WPBs) to regulate vascular function, but the underlying mechanisms are poorly understood. Here we show that histamine evokes the release of the proinflammatory ligand, P-selectin, while diverting WPBs carrying non-inflammatory cargo away from the plasma membrane to the microtubule organizing center. This differential trafficking is dependent on Rab46 (CRACR2A), a newly identified Ca2+-sensing GTPase, which localizes to a subset of P-selectin-negative WPBs. After acute stimulation of the H1 receptor, GTP-bound Rab46 evokes dynein-dependent retrograde transport of a subset of WPBs along microtubules. Upon continued histamine stimulation, Rab46 senses localized elevations of intracellular calcium and evokes dispersal of microtubule organizing center-clustered WPBs. These data demonstrate for the first time that a Rab GTPase, Rab46, integrates G protein and Ca2+ signals to couple on-demand histamine signals to selective WPB trafficking.

Homotypic endothelial nanotubes induced by wheat germ agglutinin and thrombin.

Endothelial barrier formation is maintained by intercellular communication through junctional proteins. The mechanisms involved in maintaining endothelial communication subsequent to barrier disruption remain unclear. It is known that low numbers of endothelial cells can be interconnected by homotypic actin-driven tunneling nanotubes (TNTs) which could be important for intercellular transfer of information in vascular physiology. Here we sought insight into the triggers for TNT formation. Wheat germ agglutinin, a C-type lectin and known label for TNTs, unexpectedly caused striking induction of TNTs. A succinylated derivative was by contrast inactive, suggesting mediation by a sialylated protein. Through siRNA-mediated knockdown we identified that this protein was likely to be CD31, an important sialylated membrane protein normally at endothelial cell junctions. We subsequently considered thrombin as a physiological inducer of endothelial TNTs because it reduces junctional contact. Thrombin reduced junctional contact, redistributed CD31 and induced TNTs, but its effect on TNTs was CD31-independent. Thrombin-induced TNTs nevertheless required PKCα, a known mediator of thrombin-dependent junctional remodelling, suggesting a necessity for junctional proteins in TNT formation. Indeed, TNT-inducing effects of wheat germ agglutinin and thrombin were both correlated with cortical actin rearrangement and similarly Ca2+-dependent, suggesting common underlying mechanisms. Once formed, Ca2+ signalling along TNTs was observed.

Yoda1 analogue (Dooku1) which antagonizes Yoda1-evoked activation of Piezo1 and aortic relaxation.

BACKGROUND AND PURPOSE: The mechanosensitive Piezo1 channel has important roles in vascular physiology and disease. Yoda1 is a small-molecule agonist, but the pharmacology of these channels is otherwise limited. EXPERIMENTAL APPROACH: Yoda1 analogues were generated by synthetic chemistry. Intracellular Ca2+ and Tl+ measurements were made in HEK 293 or CHO cell lines overexpressing channel subunits and in HUVECs, which natively express Piezo1. Isometric tension recordings were made from rings of mouse thoracic aorta. KEY RESULTS: Modification of the pyrazine ring of Yoda1 yielded an analogue, which lacked agonist activity but reversibly antagonized Yoda1. The analogue is referred to as Dooku1. Dooku1 inhibited 2 µM Yoda1-induced Ca2+ -entry with IC50 s of 1.3 µM (HEK 293 cells) and 1.5 µM (HUVECs) yet failed to inhibit constitutive Piezo1 channel activity. It had no effect on endogenous ATP-evoked Ca2+ elevation or store-operated Ca2+ entry in HEK 293 cells or Ca2+ entry through TRPV4 or TRPC4 channels overexpressed in CHO and HEK 293 cells. Yoda1 caused dose-dependent relaxation of aortic rings, which was mediated by an endothelium- and NO-dependent mechanism and which was antagonized by Dooku1 and analogues of Dooku1. CONCLUSION AND IMPLICATIONS: Chemical antagonism of Yoda1-evoked Piezo1 channel activity is possible, and the existence of a specific chemical interaction site is suggested with distinct binding and efficacy domains.

Piezo1 channels sense whole body physical activity to reset cardiovascular homeostasis and enhance performance.

Mammalian biology adapts to physical activity but the molecular mechanisms sensing the activity remain enigmatic. Recent studies have revealed how Piezo1 protein senses mechanical force to enable vascular development. Here, we address Piezo1 in adult endothelium, the major control site in physical activity. Mice without endothelial Piezo1 lack obvious phenotype but close inspection reveals a specific effect on endothelium-dependent relaxation in mesenteric resistance artery. Strikingly, the Piezo1 is required for elevated blood pressure during whole body physical activity but not blood pressure during inactivity. Piezo1 is responsible for flow-sensitive non-inactivating non-selective cationic channels which depolarize the membrane potential. As fluid flow increases, depolarization increases to activate voltage-gated Ca2+ channels in the adjacent vascular smooth muscle cells, causing vasoconstriction. Physical performance is compromised in mice which lack endothelial Piezo1 and there is weight loss after sustained activity. The data suggest that Piezo1 channels sense physical activity to advantageously reset vascular control.The mechanisms that regulate the body's response to exercise are poorly understood. Here, Rode et al. show that the mechanically activated cation channel Piezo1 is a molecular sensor of physical exercise in the endothelium that triggers endothelial communication to mesenteric vessel muscle cells, leading to vasoconstriction.

Upregulated WEE1 protects endothelial cells of colorectal cancer liver metastases.

Surgical resection of colorectal cancer liver metastases (CLM) can be curative, yet 80% of patients are unsuitable for this treatment. As angiogenesis is a determinant of CLM progression we isolated endothelial cells from CLM and sought a mechanism which is upregulated, essential for angiogenic properties of these cells and relevant to emerging therapeutic options. Matched CLM endothelial cells (CLMECs) and endothelial cells of normal adjacent liver (LiECs) were superficially similar but transcriptome sequencing revealed molecular differences, one of which was unexpected upregulation and functional significance of the checkpoint kinase WEE1. Western blotting confirmed that WEE1 protein was upregulated in CLMECs. Knockdown of WEE1 by targeted short interfering RNA or the WEE1 inhibitor AZD1775 suppressed proliferation and migration of CLMECs. Investigation of the underlying mechanism suggested induction of double-stranded DNA breaks due to nucleotide shortage which then led to caspase 3-dependent apoptosis. The implication for CLMEC tube formation was striking with AZD1775 inhibiting tube branch points by 83%. WEE1 inhibitors might therefore be a therapeutic option for CLM and could be considered more broadly as anti-angiogenic agents in cancer treatment.

Natural and synthetic flavonoid modulation of TRPC5 channels.

BACKGROUND AND PURPOSE: The TRPC5 proteins assemble to create calcium-permeable, non-selective, cationic channels. We sought novel modulators of these channels through studies of natural products. EXPERIMENTAL APPROACH: Intracellular calcium measurements and patch clamp recordings were made from cell lines. Compounds were generated by synthetic chemistry. KEY RESULTS: Through a screen of natural products used in traditional Chinese medicines, the flavonol galangin was identified as an inhibitor of lanthanide-evoked calcium entry in TRPC5 overexpressing HEK 293 cells (IC50 0.45 µM). Galangin also inhibited lanthanide-evoked TRPC5-mediated current in whole-cell and outside-out patch recordings. In differentiated 3T3-L1 cells, it inhibited constitutive and lanthanide-evoked calcium entry through endogenous TRPC5-containing channels. The related natural flavonols, kaempferol and quercetin were less potent inhibitors of TRPC5. Myricetin and luteolin lacked effect, and apigenin was a stimulator. Based on structure-activity relationship studies with natural and synthetic flavonols, we designed 3,5,7-trihydroxy-2-(2-bromophenyl)-4H-chromen-4-one (AM12), which inhibited lanthanide-evoked TRPC5 activity with an IC50 of 0.28 µM. AM12 also inhibited TRPC5 activity evoked by the agonist (-)-Englerin A and was effective in excised outside-out membrane patches, suggesting a relatively direct effect. It inhibited TRPC4 channels similarly, but its inhibitory effect on TRPC1-TRPC5 heteromeric channels was weaker. CONCLUSIONS AND IMPLICATIONS: The data suggest that galangin (a natural product from the ginger family) is a TRPC5 inhibitor and that other natural and synthetic flavonoids contain antagonist or agonist capabilities at TRPC5 and closely related channels depending on the substitution patterns of both the chromone core and the phenyl ring.