Melanie J. Ludlow

Piezo1 integration of vascular architecture with physiological force

The mechanisms by which physical forces regulate endothelial cells to determine the complexities of vascular structure and function are enigmatic. Studies of sensory neurons have suggested Piezo proteins as subunits of Ca2+-permeable non-selective cationic channels for detection of noxious mechanical impact. Here we show Piezo1 (Fam38a) channels as sensors of frictional force (shear stress) and determinants of vascular structure in both development and adult physiology. Global or endothelial-specific disruption of mouse Piezo1 profoundly disturbed the developing vasculature and was embryonic lethal within days of the heart beating. Haploinsufficiency was not lethal but endothelial abnormality was detected in mature vessels. The importance of Piezo1 channels as sensors of blood flow was shown by Piezo1 dependence of shear-stress-evoked ionic current and calcium influx in endothelial cells and the ability of exogenous Piezo1 to confer sensitivity to shear stress on otherwise resistant cells. Downstream of this calcium influx there was protease activation and spatial reorganization of endothelial cells to the polarity of the applied force. The data suggest that Piezo1 channels function as pivotal integrators in vascular biology.

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.

Functional Characterization of Intracellular Dictyostelium discoideum P2X Receptors

Indicative of cell surface P2X ion channel activation, extracellular ATP evokes a rapid and transient calcium influx in the model eukaryote Dictyostelium discoideum. Five P2X-like proteins (dP2XA–E) are present in this organism. However, their roles in purinergic signaling are unclear, because dP2XA proved to have an intracellular localization on the contractile vacuole where it is thought to be required for osmoregulation. To determine functional properties of the remaining four dP2X-like proteins and to assess their cellular roles, we recorded membrane currents from expressed cloned receptors and generated a quintuple knock-out Dictyostelium strain devoid of dP2X receptors. ATP evoked inward currents at dP2XB and dP2XE receptors but not at dP2XC or dP2XD. β,γ-Imido-ATP was more potent than ATP at dP2XB but a weak partial agonist at dP2XE. Currents in dP2XB and dP2XE were strongly inhibited by Na+ but insensitive to copper and the P2 receptor antagonists pyridoxal phosphate-6-azophenyl-2′,4′-disulfonic acid and suramin. Unusual for P2X channels, dP2XA and dP2XB were also Cl−-permeable. The extracellular purinergic response to ATP persisted in p2xA/B/C/D/E quintuple knock-out Dictyostelium demonstrating that dP2X channels are not responsible. dP2XB, -C, -D, and -E were found to be intracellularly localized to the contractile vacuole with the ligand binding domain facing the lumen. However, quintuple p2xA/B/C/D/E null cells were still capable of regulating cell volume in water demonstrating that, contrary to previous findings, dP2X receptors are not required for osmoregulation. Responses to the calmodulin antagonist calmidazolium, however, were reduced in p2xA/B/C/D/E null cells suggesting that dP2X receptors play a role in intracellular calcium signaling.

Purinergic-mediated Ca2+ influx in Dictyostelium discoideum

Summary The presence of five P2X-like genes (p2xA–E) in Dictyostelium suggests that nucleotides other than cAMP may act as extracellular signalling molecules in this model eukaryote. However, p2xA was found to have an exclusively intracellular localisation making it unclear whether Dictyostelium utilise P2 receptors in a manner analogous to vertebrates. Using an apoaequorin expressing strain we show here that Dictyostelium do possess cell surface P2 receptors that facilitate Ca2+ influx in response to extracellular ATP and ADP (EC50 = 7.5 μM and 6.1 μM, respectively). Indicative of P2X receptor activation, responses were rapid reaching peak within 2.91 ± 0.04 s, required extracellular Ca2+, were inhibited by Gd3+, modified by extracellular pH and were not affected by deletion of either the single Gβ or iplA genes. Responses also remained unaffected by disruption of p2xA or p2xE showing that these genes are not involved. Cu2+ and Zn2+ inhibited purine-evoked Ca2+ influx with IC50 values of 0.9 and 6.3 μM, respectively. 300 μM Zn2+ completely abolished the initial large rapid rise in intracellular Ca2+ revealing the presence of an additional smaller, slower P2Y-like response. The existence of P2 receptors in Dictyostelium makes this organism a valuable model to explore fundamental aspects of purinergic signalling.

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.

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.

(−)-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.

Natural and synthetic flavonoid modulation of TRPC5 channels

The TRPC5 proteins assemble to create calcium‐permeable, non‐selective, cationic channels. We sought novel modulators of these channels through studies of natural products.

Pico145 - powerful new tool for TRPC1/4/5 channels

The free cytosolic Ca2C concentration and voltage across the plasma membrane are major determinants of cell function. Both are regulated by Ca2C-permeable, non-selective cation channels, which are varied and numerous. Several superfamilies of protein are involved, one of which is the Transient Receptor Potential (TRP) family of membrane proteins encoded by 28 genes in mammals. TRP proteins form channels by assembling as tetramers (Fig. 1A) and heteromultimerisation (mixing together of different TRPs in a tetramer) significantly increases the number of possible TRP channels—exactly how many distinct TRP channels exist, however, remains unclear. One of the subfamilies of mammalian TRP is formed by the Canonical TRPCs, so called because they are closest in amino acid sequence to the archetypal D. melanogaster TRP. There are 7 TRPCs, all of which exist in humans except TRPC2. TRPCs are striking for their widespread expression and tendency to form heteromers. One of the heteromerising clusters consists of TRPC1, TRPC4 and TRPC5. TRPC4 and TRPC5 are each able to form homomeric channels that are biophysically similar to each other. In contrast, TRPC1 does not form homomeric channels, or does so poorly, yet it readily forms heteromers with TRPC4 and TRPC5, generating ion channels with distinct biophysical characteristics. Importantly, TRPC1 is widely expressed across many cell types, suggesting that TRPC4 and TRPC5 commonly exist in heteromers with TRPC1. However, the tools with which to determine the contributions of homomers, heteromers or combinations thereof have been lacking. TRPC1/4/5 channels seem not to have a single physiologic activator. Instead they are promiscuous— multiple modulators exist. Not all are endogenous physiologic substances; rather, some are exogenous chemicals from plants, consistent with a general idea that TRP channels help mammals integrate with their external environment. Modulators of the TRPC1/4/5 channels include receptor agonists, hydrogen peroxide, mild acidification, toxic metal ions, oxidised phospholipids, galangin and v-3 fatty acids. The most powerful, potent and selective activator is (¡)-Englerin A, generated by the East African plant Phyllanthus engleri. The physiologic significance of the (¡)-Englerin A-induced TRPC1/4/5 activation is unknown. In mice, (¡)-Englerin A is toxic. Despite limitations in tools for studying TRPC1/4/5 channels, there is encouraging evidence for roles in pathophysiology, for example in epilepsy, anxiety, pain, adverse cardiac and vascular remodelling, rheumatoid arthritis and cancer. A physiologic necessity or survival advantage of TRPC1/4/5 channels has remained unclear but suggested pro-pathophysiological roles have led to interest in the channels as targets in therapeutic drug discovery efforts. Whether the target should be TRPC1, TRPC4, TRPC5—or some combination of these—has nevertheless often been unclear. The motivation to discover and develop potent, selective small-molecule inhibitors of TRPC1/4/5 channels has therefore been 2-fold: first to facilitate basic research efforts aimed at better understanding the roles of TRPC1/4/5 channels across species, tissues

Endocytosis of HERG is clathrin-independent and involves arf6.

The hERG potassium channel is critical for repolarisation of the cardiac action potential. Reduced expression of hERG at the plasma membrane, whether caused by hereditary mutations or drugs, results in long QT syndrome and increases the risk of ventricular arrhythmias. Thus, it is of fundamental importance to understand how the density of this channel at the plasma membrane is regulated. We used antibodies to an extracellular native or engineered epitope, in conjunction with immunofluorescence and ELISA, to investigate the mechanism of hERG endocytosis in recombinant cells and validated the findings in rat neonatal cardiac myocytes. The data reveal that this channel undergoes rapid internalisation, which is inhibited by neither dynasore, an inhibitor of dynamin, nor a dominant negative construct of Rab5a, into endosomes that are largely devoid of the transferrin receptor. These results support a clathrin-independent mechanism of endocytosis and exclude involvement of dynamin-dependent caveolin and RhoA mechanisms. In agreement, internalised hERG displayed marked overlap with glycosylphosphatidylinositol-anchored GFP, a clathrin-independent cargo. Endocytosis was significantly affected by cholesterol extraction with methyl-β-cyclodextrin and inhibition of Arf6 function with dominant negative Arf6-T27N-eGFP. Taken together, we conclude that hERG undergoes clathrin-independent endocytosis via a mechanism involving Arf6.