Remarkable Progress with Small-Molecule Modulation of TRPC1/4/5 Channels: Implications for Understanding the Channels in Health and Disease

Proteins of the TRPC family can form many homo- and heterotetrameric cation channels permeable to Na+, K+ and Ca2+. In this review, we focus on channels formed by the isoforms TRPC1, TRPC4 and TRPC5. We review evidence for the formation of different TRPC1/4/5 tetramers, give an overview of recently developed small-molecule TRPC1/4/5 activators and inhibitors, highlight examples of biological roles of TRPC1/4/5 channels in different tissues and pathologies, and discuss how high-quality chemical probes of TRPC1/4/5 modulators can be used to understand the involvement of TRPC1/4/5 channels in physiological and pathophysiological processes

Piezo1 channel activation mimics high glucose as a stimulator of insulin release

Glucose and hypotonicity induced cell swelling stimulate insulin release from pancreatic β-cells but the mechanisms are poorly understood. Recently, Piezo1 was identified as a mechanically-activated nonselective Ca2+ permeable cationic channel in a range of mammalian cells. As cell swelling induced insulin release could be through stimulation of Ca2+ permeable stretch activated channels, we hypothesised a role for Piezo1 in cell swelling induced insulin release. Two rat β-cell lines (INS-1 and BRIN-BD11) and freshly-isolated mouse pancreatic islets were studied. Intracellular Ca2+ measurements were performed using the fura-2 Ca2+ indicator dye and ionic current was recorded by whole cell patch-clamp. Piezo1 agonist Yoda1, a competitive antagonist of Yoda1 (Dooku1) and an inactive analogue of Yoda1 (2e) were used as chemical probes. Piezo1 mRNA and insulin secretion were measured by RT-PCR and ELISA respectively. Piezo1 mRNA was detected in both β-cell lines and mouse islets. Yoda1 evoked Ca2+ entry was inhibited by Yoda1 antagonist Dooku1 as well as other Piezo1 inhibitors gadolinium and ruthenium red, and not mimicked by 2e. Yoda1, but not 2e, stimulated Dooku1-sensitive insulin release from β-cells and pancreatic islets. Hypotonicity and high glucose increased intracellular Ca2+ and enhanced Yoda1 Ca2+ influx responses. Yoda1 and hypotonicity induced insulin release were significantly inhibited by Piezo1 specific siRNA. Pancreatic islets from mice with haploinsufficiency of Piezo1 released less insulin upon exposure to Yoda1. The data show that Piezo1 channel agonist induces insulin release from β-cell lines and mouse pancreatic islets suggesting a role for Piezo1 in cell swelling induced insulin release. Hence Piezo1 agonists have the potential to be used as enhancers of insulin release

Mechanically activated Piezo1 channels of cardiac fibroblasts stimulate p38 mitogen-activated protein kinase activity and interleukin-6 secretion

Piezo1 is a mechanosensitive cation channel with widespread physiological importance; however, its role in the heart is poorly understood. Cardiac fibroblasts help preserve myocardial integrity and play a key role in regulating its repair and remodeling following stress or injury. Here we investigated Piezo1 expression and function in cultured human and mouse cardiac fibroblasts. RT-PCR experiments confirmed that Piezo1 mRNA in cardiac fibroblasts is expressed at levels similar to those in endothelial cells. The results of a Fura-2 intracellular Ca2+ assay validated Piezo1 as a functional ion channel that is activated by its agonist, Yoda1. Yoda1-induced Ca2+ entry was inhibited by Piezo1 blockers (gadolinium and ruthenium red) and was reduced proportionally by siRNA-mediated Piezo1 knockdown or in murine Piezo1+/− cells. Results from cell-attached patch clamp recordings on human cardiac fibroblasts established that they contain mechanically activated ion channels and that their pressure responses are reduced by Piezo1 knockdown. Investigation of Yoda1 effects on selected remodeling genes indicated that Piezo1 activation increases both mRNA levels and protein secretion of IL-6, a pro-hypertrophic and profibrotic cytokine, in a Piezo1-dependent manner. Moreover, Piezo1 knockdown reduced basal IL-6 expression from cells cultured on softer collagen-coated substrates. Multiplex kinase activity profiling combined with kinase inhibitor experiments and phosphospecific immunoblotting established that Piezo1 activation stimulates IL-6 secretion via the p38 mitogen-activated protein kinase downstream of Ca2+ entry. In summary, cardiac fibroblasts express mechanically activated Piezo1 channels coupled to secretion of the paracrine signaling molecule IL-6. Piezo1 may therefore be important in regulating cardiac remodeling

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

Changes in Lysozyme Flexibility upon Mutation Are Frequent, Large and Long-Ranged

We investigate changes in human c-type lysozyme flexibility upon mutation via a Distance Constraint Model, which gives a statistical mechanical treatment of network rigidity. Specifically, two dynamical metrics are tracked. Changes in flexibility index quantify differences within backbone flexibility, whereas changes in the cooperativity correlation quantify differences within pairwise mechanical couplings. Regardless of metric, the same general conclusions are drawn. That is, small structural perturbations introduced by single point mutations have a frequent and pronounced affect on lysozyme flexibility that can extend over long distances. Specifically, an appreciable change occurs in backbone flexibility for 48% of the residues, and a change in cooperativity occurs in 42% of residue pairs. The average distance from mutation to a site with a change in flexibility is 17–20 Å. Interestingly, the frequency and scale of the changes within single point mutant structures are generally larger than those observed in the hen egg white lysozyme (HEWL) ortholog, which shares 61% sequence identity with human lysozyme. For example, point mutations often lead to substantial flexibility increases within the β-subdomain, which is consistent with experimental results indicating that it is the nucleation site for amyloid formation. However, β-subdomain flexibility within the human and HEWL orthologs is more similar despite the lowered sequence identity. These results suggest compensating mutations in HEWL reestablish desired properties

Reliability and validity of ultrasound imaging of features of knee osteoarthritis in the community

<p>Abstract</p> <p>Background</p> <p>Radiographs are the main outcome measure in epidemiological studies of osteoarthritis (OA). Ultrasound imaging has unique advantages in that it involves no ionising radiation, is easy to use and visualises soft tissue structures. Our objective was to measure the inter-rater reliability and validity of ultrasound imaging in the detection of features of knee OA.</p> <p>Methods</p> <p>Eighteen participants from a community cohort, had both knees scanned by two trained musculoskeletal sonographers, up to six weeks apart. Inter-rater reliability for osteophytes, effusion size and cartilage thickness was calculated by estimating Kappa (κ) and Intraclass correlation coefficients (ICC), as appropriate. A measure of construct validity was determined by estimating κ between the two imaging modalities in the detection of osteophytes.</p> <p>Results</p> <p><it>Reliability: </it>κ for osteophyte presence was 0.77(right femur), 0.65(left femur) and 0.88 for both tibia. ICCs for effusion size were 0.70(right) and 0.85(left). Moderate to substantial agreement was found in cartilage thickness measurements. <it>Validity: </it>For osteophytes, κ was moderate to excellent at 0.52(right) and 0.75(left).</p> <p>Conclusion</p> <p>Substantial to excellent agreement was found between ultrasound observers for the presence of osteophytes and measurement of effusion size; it was moderate to substantial for femoral cartilage thickness. Moderate to substantial agreement was observed between ultrasound and radiographs for osteophyte presence.</p

Evolutionary and pulsational properties of white dwarf stars

Abridged. White dwarf stars are the final evolutionary stage of the vast majority of stars, including our Sun. The study of white dwarfs has potential applications to different fields of astrophysics. In particular, they can be used as independent reliable cosmic clocks, and can also provide valuable information about the fundamental parameters of a wide variety of stellar populations, like our Galaxy and open and globular clusters. In addition, the high densities and temperatures characterizing white dwarfs allow to use these stars as cosmic laboratories for studying physical processes under extreme conditions that cannot be achieved in terrestrial laboratories. They can be used to constrain fundamental properties of elementary particles such as axions and neutrinos, and to study problems related to the variation of fundamental constants. In this work, we review the essentials of the physics of white dwarf stars. Special emphasis is placed on the physical processes that lead to the formation of white dwarfs as well as on the different energy sources and processes responsible for chemical abundance changes that occur along their evolution. Moreover, in the course of their lives, white dwarfs cross different pulsational instability strips. The existence of these instability strips provides astronomers with an unique opportunity to peer into their internal structure that would otherwise remain hidden from observers. We will show that this allows to measure with unprecedented precision the stellar masses and to infer their envelope thicknesses, to probe the core chemical stratification, and to detect rotation rates and magnetic fields. Consequently, in this work, we also review the pulsational properties of white dwarfs and the most recent applications of white dwarf asteroseismology.Comment: 85 pages, 28 figures. To be published in The Astronomy and Astrophysics Revie