The mechanisms by which physical forces regulate endothelial cells to determine the complexities of vascular structure and function are enigmatic1-5. Studies of sensory neurons have suggested Piezo proteins as subunits of Ca2+-permeable non-selective cationic channels for detection of noxious mechanical impact6-8. 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. 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 was protease activity and spatial organization of endothelial cells to the polarity of the applied force. The data suggest Piezo1 channels as pivotal integrators in vascular biology.
Rationale: Orai1 and the associated calcium release-activated calcium (CRAC) channel were discovered in the immune system. Existence also in endothelial cells has been suggested, but the relevance to endothelial biology is mostly unknown.Objective: The aim of this study was to investigate the relevance of Orai1 and CRAC channels to vascular endothelial growth factor (VEGF) and endothelial tube formation. Methods and Results:
Mammalian homologues of Drosophila melanogaster transient receptor potential (TRP) are a large family of multimeric cation channels that act, or putatively act, as sensors of one or more chemical factor1,2. Major research objectives are the identification of endogenous activators and the determination of cellular and tissue functions of these novel channels. Here we show activation of TRPC5 homomultimeric and TRPC5-TRPC1 heteromultimeric channels3-5 by extracellular reduced thioredoxin acting by breaking a disulphide bridge in the predicted extracellular loop adjacent to the ion-selectivity filter of TRPC5. Thioredoxin is an endogenous redox protein with established intracellular functions, but it is also secreted and its extracellular targets are largely unknown6-9. Particularly high extracellular concentrations of thioredoxin are apparent in rheumatoid arthritis8,10-12, an inflammatory joint disease disabling millions of people worldwide13. We show that TRPC5 and TRPC1 are expressed in secretory fibroblast-like synoviocytes from patients with rheumatoid arthritis, endogenous TRPC5-TRPC1 channels of the cells are activated by reduced thioredoxin, and blockade of the channels enhances secretory activity and prevents suppression of secretion by thioredoxin. The data suggest a novel ion channel activation mechanism that couples extracellular thioredoxin to cell function.Striking activators of TRPC5 are extracellular lanthanide ions4,14,15. Effects of these ions depend on a glutamic acid residue at position 54314 in the predicted extracellular loop adjacent to the ion pore (Supplementary Fig. 1-2). This structural feature may, therefore, have functional importance in enabling extracellular factors to activate the channels. Because lanthanides are unlikely physiological activators we were interested in alternatives and developed a hypothesis based on amino acid sequence alignment which showed two cysteine residues near glutamic acid 543 that are conserved in TRPC5, TRPC4 and TRPC1 ( Supplementary Fig. 2), a subset of the seven TRPC channels1-5. TRPC5 and TRPC4 have similar functional properties4 and both form heteromultimers with TRPC13-5, a subunit that has weak targeting to the plasma membrane when expressed in isolation3,16. Pairs of cysteine residues may be covalently linked by a disulphide bridge that can be cleaved by reduction. We therefore applied the chemical reducing agent dithiothreitol (DTT) to HEK 293 cells expressing TRPC515,16. There was channel activation with the characteristic current-voltage relationship (I-V) of TRPC5 and block by 2-APB, an inhibitor of TRPC55 (Fig. 1a, b, d). Current recovered on wash-out of DTT (data not shown). Similarly, the membrane-impermeable disulphide reducing agent TCEP (Fig. 1c, d) activated TRPC5, whereas the thiol reagent MTSET had no effect (Fig. 1d). TRPC5 was inhibited by cadmium ions only after pre-treatment with DTT ( Fig. 1e, f), consistent with the metal ion acting by re-engaging cysteines17. Other TRP channels lacking the cysteine pair in a similar po...
The channels are thought to have structural similarity to ␣-subunits of voltage-gated K ϩ channels, with intracellular amino and carboxy termini and four proteins required for coordination of a single ion pore. As with K ϩ channels, heteromultimerization confers greater diversity. However, unlike voltage-gated K ϩ channels, membrane depolarization is not the primary trigger for channel activity. Instead, chemical factors are considered to be primary stimuli. Details of the chemical sensing properties are becoming apparent and hold promise for revealing further complexity and novelty. In addition, important roles of TRP channels have emerged, including in sensation and cell survival, but we are far from a full appreciation of the purposes of these channels and, in some cases, there is relatively little understanding of TRP family members -one example being TRPM3.
Robotic multiwell planar patch-clamp has become common in drug development and safety programs because it enables efficient and systematic testing of compounds against ion channels during voltage-clamp. It has not, however, been adopted significantly in other important areas of ion channel research, where conventional patch-clamp remains the favored method. Here, we show the wider potential of the multiwell approach with the ability for efficient intracellular solution exchange, describing protocols and success rates for recording from a range of native and primary mammalian cells derived from blood vessels, arthritic joints and the immune and central nervous systems. The protocol involves preparing a suspension of single cells to be dispensed robotically into 4-8 microfluidic chambers each containing a glass chip with a small aperture. Under automated control, giga-seals and whole-cell access are achieved followed by preprogrammed routines of voltage paradigms and fast extracellular or intracellular solution exchange. Recording from 48 chambers usually takes 1-6 h depending on the experimental design and yields 16-33 cell recordings.
BACKGROUND AND PURPOSE The transient receptor potential melastatin‐3 (TRPM3) channel forms calcium‐permeable, non‐selective, cationic channels that are stimulated by pregnenolone sulphate (PregS). Here, we aimed to define chemical requirements of this acute steroid action and potentially reveal novel stimulators with physiological relevance. EXPERIMENTAL APPROACH We used TRPM3 channels over‐expressed in HEK 293 cells, with intracellular calcium measurement and whole‐cell patch‐clamp recording techniques. KEY RESULTS The stimulation of TRPM3 channels was confined to PregS and closely related steroids and not mimicked by other major classes of steroids, including progesterone. Relatively potent stimulation of TRPM3‐dependent calcium entry was observed. A sulphate group positioned at ring A was important for strong stimulation but more striking was the requirement for a cis (β) configuration of the side group, revealing previously unrecognized stereo‐selectivity and supporting existence of a specific binding site. A cis‐oriented side group on ring A was not the only feature necessary for high activity because loss of the double bond in ring B reduced potency and loss of the acetyl group at ring D reduced efficacy and potency. Weak steroid stimulators of TRPM3 channels inhibited effects of PregS, suggesting partial agonism. In silico screening of chemical libraries for non‐steroid modulators of TRPM3 channels revealed the importance of the steroid backbone for stimulatory effects. CONCLUSIONS AND IMPLICATIONS Our data defined some of the chemical requirements for acute stimulation of TRPM3 channels by steroids, supporting the existence of a specific and unique steroid binding site. Epipregnanolone sulphate was identified as a novel TRPM3 channel stimulator.
Transient Receptor Potential Melastatin 3 (TRPM3) is a widely expressed calcium-permeable non-selective cation channel that is stimulated by high concentrations of nifedipine or by physiological steroids that include pregnenolone sulphate. Here we sought to identify steroids that inhibit TRPM3. Channel activity was studied using calcium-measurement and patch-clamp techniques. Progesterone (0.01–10 μM) suppressed TRPM3 activity evoked by pregnenolone sulphate. Progesterone metabolites and 17β-oestradiol were also inhibitory but the effects were relatively small. Dihydrotestosterone was an inhibitor at concentrations higher than 1 μM. Corticosteroids lacked effect. Overlay assays indicated that pregnenolone sulphate, progesterone and dihydrotestosterone bound to TRPM3. In contrast to dihydrotestosterone, progesterone inhibited nifedipine-evoked TRPM3 activity or activity in the absence of an exogenous activator, suggesting a pregnenolone sulphate-independent mechanism of action. Dihydrotestosterone, like a non-steroid look-alike compound, acted as a competitive antagonist at the pregnenolone sulphate binding site. Progesterone inhibited endogenous TRPM3 in vascular smooth muscle cells. Relevance of TRPM3 or the progesterone effect to ovarian cells, which have been suggested to express TRPM3, was not identified. The data further define a chemical framework for competition with pregnenolone sulphate at TRPM3 and expand knowledge of steroid interactions with TRPM3, suggesting direct steroid binding and pregnenolone sulphate-independent inhibition by progesterone.
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