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

The concentration of free cytosolic Ca(2+) and the voltage across the plasma membrane are major determinants of cell function. Ca(2+)-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 Ca(2+)-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 which are high-quality, reliable, easy to use and readily accessible for all investigators. Here, through chemical synthesis and studies of native and over-expressed channels by Ca(2+) and patch-clamp assays, we describe compound 31 (C31), 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 Ca(2+) 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

From Aristotle to Arendt : a phenomenological exploration of forms of knowledge and practice in the context of child protection social work in the UK

This paper attempts to explore the relationship between different forms of knowledge and the kinds of activity that arise from them within child protection social work practice. The argument that social work is more than either ‘science’ or ‘art’ but distinctly ‘practice’ is put through a historical description of the development of Aristotle’s views of the forms of knowledge and Hannah Arendt’s later conceptualisations as detailed in The Human Condition (1958). The paper supports Arendt’s privileging of Praxis over Theoria within social work and further draws upon Arendt’s distinctions between Labour, Work and Action to delineate between different forms of social work activity. The author highlights dangers in social work relying too heavily on technical knowledge and the use of theory as a tool in seeking to understand and engage with the people it serves and stresses the importance of a phenomenological approach to research and practice as a valid, embodied form of knowledge. The argument further explores the constructions of service users that potentially arise from different forms of social work activity and cautions against over-prescriptive use of ‘outcomes’ based practice that may reduce the people who use services to products or consumables. The author concludes that social work action inevitably involves trying to understand humans in a complex and dynamic way that requires engagement and to seek new meanings for individual humans

Orai1 Channel Inhibition Preserves Left Ventricular Systolic Function and Normal Ca2+ Handling After Pressure Overload

Background: Orai1 is a critical ion channel subunit, best recognized as a mediator of storeoperated Ca2+ entry (SOCE) in non-excitable cells. SOCE has recently emerged as a key contributor of cardiac hypertrophy and heart failure but the relevance of Orai1 is still unclear. Methods: To test the role of these Orai1 channels in the cardiac pathophysiology, a transgenic mouse was generated with cardiomyocyte-specific expression of an ion pore-disruptive Orai1R91W mutant (C-dnO1). Synthetic chemistry and channel screening strategies were used to develop JPIII, a small-molecule Orai1 channel inhibitor suitable for in vivo delivery. Results: Adult mice subjected to transverse aortic constriction (TAC) developed cardiac hypertrophy and reduced ventricular function associated with increased Orai1 expression and Orai1-dependent SOCE (assessed by Mn2+ influx). C-dnO1 mice displayed normal cardiac electromechanical function and cellular excitation-contraction coupling despite reduced Orai1-dependent SOCE. 5 weeks after TAC, C-dnO1 mice were protected from systolic dysfunction (assessed by preserved left ventricular fractional shortening and ejection fraction) even if increased cardiac mass and pro-hypertrophic markers induction were observed. This is correlated with a protection from TAC-induced cellular Ca2+ signaling alterations (increased SOCE, decreased [Ca2+]i transients amplitude and decay rate, lower SR Ca2+ load and depressed cellular contractility) and SERCA2a downregulation in ventricular cardiomyocytes from C-dnO1 mice, associated with blunted Pyk2 signaling. There was also less fibrosis in heart sections from CdnO1 mice after TAC. Moreover, 3 weeks treatment with JPIII following 5 weeks of TAC confirmed the translational relevance of an Orai1 inhibition strategy during hypertrophic insult. Conclusions: The findings suggest a key role of cardiac Orai1 channels and the potential for Orai1 channel inhibitors as inotropic therapies for maintaining contractility reserve after hypertrophic stress

Orai3 Surface Accumulation and Calcium Entry Evoked by Vascular Endothelial Growth Factor

Objective: Vascular endothelial growth factor (VEGF) acts, in part, by triggering calcium ion (Ca2+) entry. Here, we sought understanding of a Synta66-resistant Ca2+ entry pathway activated by VEGF. Approach and Results: Measurement of intracellular Ca2+ in human umbilical vein endothelial cells detected a Synta66-resistant component of VEGF-activated Ca2+ entry that occurred within 2 minutes after VEGF exposure. Knockdown of the channel-forming protein Orai3 suppressed this Ca2+ entry. Similar effects occurred in 3 further types of human endothelial cell. Orai3 knockdown was inhibitory for VEGF-dependent endothelial tube formation in Matrigel in vitro and in vivo in the mouse. Unexpectedly, immunofluorescence and biotinylation experiments showed that Orai3 was not at the surface membrane unless VEGF was applied, after which it accumulated in the membrane within 2 minutes. The signaling pathway coupling VEGF to the effect on Orai3 involved activation of phospholipase Cγ1, Ca2+ release, cytosolic group IV phospholipase A2α, arachidonic acid production, and, in part, microsomal glutathione S-transferase 2, an enzyme which catalyses the formation of leukotriene C4 from arachidonic acid. Shear stress reduced microsomal glutathione S-transferase 2 expression while inducing expression of leukotriene C4 synthase, suggesting reciprocal regulation of leukotriene C4–synthesizing enzymes and greater role of microsomal glutathione S-transferase 2 in low shear stress. Conclusions: VEGF signaling via arachidonic acid and arachidonic acid metabolism causes Orai3 to accumulate at the cell surface to mediate Ca2+ entry and downstream endothelial cell remodeling