The activation mechanism of the classical transient receptor potential channels TRPC4 and -5 via the Gq/11 protein-phospholipase C (PLC) signaling pathway has remained elusive so far. In contrast to ...all other TRPC channels, the PLC product diacylglycerol (DAG) is not sufficient for channel activation, whereas TRPC4/5 channel activity is potentiated by phosphatidylinositol 4,5-bisphosphate (PIP₂) depletion. As a characteristic structural feature, TRPC4/5 channels contain a C-terminal PDZ-binding motif allowing for binding of the scaffolding proteins Na⁺/H⁺ exchanger regulatory factor (NHERF) 1 and 2. PKC inhibition or the exchange of threonine for alanine in the C-terminal PDZ-binding motif conferred DAG sensitivity to the channel. Altogether, we present a DAG-mediated activation mechanism for TRPC4/5 channels tightly regulated by NHERF1/2 interaction. PIP₂ depletion evokes a C-terminal conformational change of TRPC5 proteins leading to dynamic dissociation of NHERF1/2 from the C terminus of TRPC5 as a prerequisite for DAG sensitivity. We show that NHERF proteins are direct regulators of ion channel activity and that DAG sensitivity is a distinctive hallmark of TRPC channels.
G protein-mediated stretch reception Storch, Ursula; Mederos y Schnitzler, Michael; Gudermann, Thomas
American journal of physiology. Heart and circulatory physiology,
2012-Mar-15, Letnik:
302, Številka:
6
Journal Article
Recenzirano
Mechanosensation and -transduction are important for physiological processes like the senses of touch, hearing, and balance. The mechanisms underlying the translation of mechanical stimuli into ...biochemical information by activating various signaling pathways play a fundamental role in physiology and pathophysiology but are only poorly understood. Recently, G protein-coupled receptors (GPCRs), which are essential for the conversion of light, olfactory and gustatory stimuli, as well as of primary messengers like hormones and neurotransmitters into cellular signals and which play distinct roles in inflammation, cell growth, and differentiation, have emerged as potential mechanosensors. The first candidate for a mechanosensitive GPCR was the angiotensin-II type-1 (AT(1)) receptor. Agonist-independent mechanical receptor activation of AT(1) receptors induces an active receptor conformation that appears to differ from agonist-induced receptor conformations and entails the activation of G proteins. Mechanically induced AT(1) receptor activation plays an important role for myogenic vasoconstriction and for the initiation of cardiac hypertrophy. A growing body of evidence suggests that other GPCRs are involved in mechanosensation as well. These findings highlight physiologically relevant, ligand-independent functions of GPCRs and add yet another facet to the polymodal activation spectrum of this ubiquitous protein family.
Despite the central physiological function of the myogenic response, the underlying signalling pathways and the identity of mechanosensors in vascular smooth muscle (VSM) are still elusive. In ...contrast to present thinking, we show that membrane stretch does not primarily gate mechanosensitive transient receptor potential (TRP) ion channels, but leads to agonist‐independent activation of Gq/11‐coupled receptors, which subsequently signal to TRPC channels in a G protein‐ and phospholipase C‐dependent manner. Mechanically activated receptors adopt an active conformation, allowing for productive G protein coupling and recruitment of β‐arrestin. Agonist‐independent receptor activation by mechanical stimuli is blocked by specific antagonists and inverse agonists. Increasing the AT1 angiotensin II receptor density in mechanically unresponsive rat aortic A7r5 cells resulted in mechanosensitivity. Myogenic tone of cerebral and renal arteries is profoundly diminished by the inverse angiotensin II AT1 receptor agonist losartan independently of angiotensin II (AII) secretion. This inhibitory effect is enhanced in blood vessels of mice deficient in the regulator of G‐protein signalling‐2. These findings suggest that Gq/11‐coupled receptors function as sensors of membrane stretch in VSM cells.
Regional alveolar hypoxia causes local vasoconstriction in the lung, shifting blood flow from hypoxic to normoxic areas, thereby maintaining gas exchange. This mechanism is known as hypoxic pulmonary ...vasoconstriction (HPV). Disturbances in HPV can cause life-threatening hypoxemia whereas chronic hypoxia triggers lung vascular remodeling and pulmonary hypertension. The signaling cascade of this vitally important mechanism is still unresolved. Using transient receptor potential channel 6 (TRPC6)-deficient mice, we show that this channel is a key regulator of acute HPV as this regulatory mechanism was absent in $TRPC6^{-/-}$ mice whereas the pulmonary vasoconstrictor response to the thromboxane mimetic U46619 was unchanged. Accordingly, induction of regional hypoventilation resulted in severe arterial hypoxemia in $TRPC6^{-/-}$ but not in WT mice. This effect was mirrored by a lack of hypoxiainduced cation influx and currents in smooth-muscle cells from precapillary pulmonary arteries (PASMC) of $TRPC6^{-/-}$ mice. In both WT and $TRPC6^{-/-}$ PASMC hypoxia caused diacylglycerol (DAG) accumulation. DAG seems to exert its action via TRPC6, as DAG kinase inhibition provoked a cation influx only in WT but not in $TRPC6^{-/-}$ PASMC. Notably, chronic hypoxia-induced pulmonary hypertension was independent of TRPC6 activity. We conclude that TRPC6 plays a unique and indispensable role in acute hypoxic pulmonary vasoconstriction. Manipulation of TRPC6 function may thus offer a therapeutic strategy for the control of pulmonary hemodynamics and gas exchange.
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•The cryo-EM structures of the human TRPC5 channel reveal two distinct drug binding sites.•TRPC5 channels possess distinct binding sites for benzimidazole and methylxanthine ...derivatives.•TRPC5 channels exhibit a binding site for the second messenger diacylglycerol near the pore region.
The transient receptor potential classical or canonical (TRPC) 5 channel is a non-selective calcium-permeable cation channel that recently emerged as a promising target for the treatment of various diseases such as mental disorders and kidney diseases. Thus, detailed insight into the structural properties of TRPC5 channels is of utmost importance to further advance TRPC5 channels as drug targets. Recently, Song et al. (2021) have presented cryo-EM structures of the human TRPC5 channel alone or in complex with two different inhibitors thereby revealing two new distinct drug binding sites. Moreover, a binding site for the second messenger diacylglycerol (DAG) has been identified commensurate with a key role of DAG for TRPC5 channel activation.
Lung ischaemia-reperfusion-induced oedema (LIRE) is a life-threatening condition that causes pulmonary oedema induced by endothelial dysfunction. Here we show that lungs from mice lacking ...nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (Nox2(y/-)) or the classical transient receptor potential channel 6 (TRPC6(-/-)) are protected from LIR-induced oedema (LIRE). Generation of chimeric mice by bone marrow cell transplantation and endothelial-specific Nox2 deletion showed that endothelial Nox2, but not leukocytic Nox2 or TRPC6, are responsible for LIRE. Lung endothelial cells from Nox2- or TRPC6-deficient mice showed attenuated ischaemia-induced Ca(2+) influx, cellular shape changes and impaired barrier function. Production of reactive oxygen species was completely abolished in Nox2(y/-) cells. A novel mechanistic model comprising endothelial Nox2-derived production of superoxide, activation of phospholipase C-γ, inhibition of diacylglycerol (DAG) kinase, DAG-mediated activation of TRPC6 and ensuing LIRE is supported by pharmacological and molecular evidence. This mechanism highlights novel pharmacological targets for the treatment of LIRE.
Platelets are known to play a crucial role in hemostasis. Sphingosine kinases (Sphk) 1 and 2 catalyze the conversion of sphingosine to the bioactive metabolite sphingosine 1-phosphate (S1P). Although ...platelets are able to secrete S1P on activation, little is known about a potential intrinsic effect of S1P on platelet function.
To investigate the role of Sphk1- and Sphk2-derived S1P in the regulation of platelet function.
We found a 100-fold reduction in intracellular S1P levels in platelets derived from Sphk2(-/-) mutants compared with Sphk1(-/-) or wild-type mice, as analyzed by mass spectrometry. Sphk2(-/-) platelets also failed to secrete S1P on stimulation. Blood from Sphk2-deficient mice showed decreased aggregation after protease-activated receptor 4-peptide and adenosine diphosphate stimulation in vitro, as assessed by whole blood impedance aggregometry. We revealed that S1P controls platelet aggregation via the sphingosine 1-phosphate receptor 1 through modulation of protease-activated receptor 4-peptide and adenosine diphosphate-induced platelet activation. Finally, we show by intravital microscopy that defective platelet aggregation in Sphk2-deficient mice translates into reduced arterial thrombus stability in vivo.
We demonstrate that Sphk2 is the major Sphk isoform responsible for the generation of S1P in platelets and plays a pivotal intrinsic role in the control of platelet activation. Correspondingly, Sphk2-deficient mice are protected from arterial thrombosis after vascular injury, but have normal bleeding times. Targeting this pathway could therefore present a new therapeutic strategy to prevent thrombosis.
Myogenic vasoconstriction is mediated by vascular smooth muscle cells of resistance arteries sensing mechanical stretch. Angiotensin II AT1 receptors and in particular AT1BRs in murine vascular ...smooth muscle cells have been characterized as mechanosensors that cannot fully account for myogenic vasoconstriction observed. Therefore, we aimed at uncovering novel vascular mechanosensors by expression profiling and functional characterization of candidate proteins.
Analyzing myogenic tone of isolated murine mesenteric arteries of AT1A and AT1B receptor double gene-deficient (AT1A/1B (-/-)) mice ex vivo, we observed a decreased myogenic tone at high intraluminal pressures and an unexpected hyper-reactivity at low intraluminal pressures because of upregulation of cysteinyl leukotriene 1 receptors (CysLT1Rs). Pharmacological blockade of CysLT1Rs with pranlukast significantly reduced myogenic tone not only in AT1A/1B (-/-) but also in wild-type arteries. In wild-type arteries, additional blockade of angiotensin II AT1 receptors with candesartan resulted in an additive reduction of myogenic tone. Furthermore, calcium imaging experiments were performed with fura-2-loaded human embryonic kidney 293 cells overexpressing CysLT1Rs and with isolated mesenteric vascular smooth muscle cells. Hypo-osmotically induced membrane stretch provoked calcium transients that were significantly reduced by pranlukast. Incubations of isolated mesenteric vascular smooth muscle cells with the 5-lipoxygenase inhibitor zileuton had no effect. Furthermore, the Gq/11-protein inhibitor YM 254890 profoundly reduced myogenic tone to the same extent as induced by the application of pranlukast plus candesartan.
Here, we identify a novel, hitherto unappreciated role of CysLT1Rs in vascular regulation. We identified CysLT1Rs as novel mechanosensors in the vasculature involved in myogenic vasoconstriction. Moreover, our findings suggest that myogenic tone is determined by AT1 and CysLT1 receptors acting together as mechanosensors via Gq/11-protein activation.
Molecular shape and pharmacological function are interconnected. To capture shape, the fractal dimensionality concept was employed, providing a natural similarity measure for the virtual screening of ...de novo generated small molecules mimicking the structurally complex natural product (−)‐englerin A. Two of the top‐ranking designs were synthesized and tested for their ability to modulate transient receptor potential (TRP) cation channels which are cellular targets of (−)‐englerin A. Intracellular calcium assays and electrophysiological whole‐cell measurements of TRPC4 and TRPM8 channels revealed potent inhibitory effects of one of the computer‐generated compounds. Four derivatives of this identified hit compound had comparable effects on TRPC4 and TRPM8. The results of this study corroborate the use of fractal dimensionality as an innovative shape‐based molecular representation for molecular scaffold‐hopping.
The shape of molecules can be described in terms of their fractal dimension, a measure of the ‘complexity’ of the molecular surface. A set of de novo designs was ranked according to fractal dimension similarity to the natural product template (−)‐englerin A. One high ranked compound potently blocked TRPC4 and TRPM8 ion channels. The study shows the potential of fractal dimensionality as a molecular shape representation for virtual screening, scaffold‐hopping, and ligand‐based drug discovery.
Impaired magnesium reabsorption in patients with TRPM6 gene mutations stresses an important role of TRPM6 (melastatin-related TRP cation channel) in epithelial magnesium transport. While attempting ...to isolate full-length TRPM6, we found that the human TRPM6 gene encodes multiple mRNA isoforms. Full-length TRPM6 variants failed to form functional channel complexes because they were retained intracellularly on heterologous expression in HEK 293 cells and Xenopus oocytes. However, TRPM6 specifically interacted with its closest homolog, the Mg2+-permeable cation channel TRPM7, resulting in the assembly of functional TRPM6/TRPM7 complexes at the cell surface. The naturally occurring S141L TRPM6 missense mutation abrogated the oligomeric assembly of TRPM6, thus providing a cell biological explanation for the human disease. Together, our data suggest an important contribution of TRPM6/TRPM7 heterooligomerization for the biological role of TRPM6 in epithelial magnesium absorption.
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