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Lutz Birnbaumer - One of the best experts on this subject based on the ideXlab platform.
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trpc1 and trpc3 dependent ca2 signaling in mouse cortical astrocytes affects injury evoked astrogliosis in vivo
Glia, 2017Co-Authors: Thabet Belkacemi, Lutz Birnbaumer, Alexander Niermann, Laura Hofmann, Ulrich Wissenbach, Petra Leidinger, Christina Backes, Eckart Meese, Andreas KellerAbstract:Following brain injury astrocytes change into a reactive state, proliferate and grow into the site of lesion, a process called astrogliosis, initiated and regulated by changes in cytoplasmic Ca2+ . Transient receptor potential canonical (TRPC) channels may contribute to Ca2+ influx but their presence and possible function in astrocytes is not known. By RT-PCR and RNA sequencing we identified transcripts of Trpc1, TRPC2, Trpc3, and Trpc4 in FACS-sorted glutamate aspartate transporter (GLAST)-positive cultured mouse cortical astrocytes and subcloned full-length Trpc1 and Trpc3 cDNAs from these cells. Ca2+ entry in cortical astrocytes depended on TRPC3 and was increased in the absence of Trpc1. After co-expression of Trpc1 and Trpc3 in HEK-293 cells both proteins co-immunoprecipitate and form functional heteromeric channels, with TRPC1 reducing TRPC3 activity. In vitro, lack of Trpc3 reduced astrocyte proliferation and migration whereas the TRPC3 gain-of-function moonwalker mutation and Trpc1 deficiency increased astrocyte migration. In vivo, astrogliosis and cortex edema following stab wound injury were reduced in Trpc3-/- but increased in Trpc1-/- mice. In summary, our results show a decisive contribution of TRPC3 to astrocyte Ca2+ signaling, which is even augmented in the absence of Trpc1, in particular following brain injury. Targeted therapies to reduce TRPC3 channel activity in astrocytes might therefore be beneficial in traumatic brain injury.
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deletion of diacylglycerol responsive trpc genes attenuates diabetic nephropathy by inhibiting activation of the tgfβ1 signaling pathway
American Journal of Translational Research, 2017Co-Authors: Benju Liu, Lutz Birnbaumer, Yanhong LiaoAbstract:TRPC6 plays a critical role in proteinuric kidney diseases, and TRPC3 is involved in tubulointerstitial damage and renal fibrosis in obstructed kidneys. Podocyte loss is a characteristic event in diabetic nephropathy (DN). The aim of this study was to examine whether deletion of the closely related diacylglycerol (DAG)-responsive TRPCs in mice (TRPC3/6/7-/-) affects diabetes-induced renal dysfunction and podocyte loss. We compared urine volume, kidney hypertrophy, glomerular enlargement, albuminuria and podocyte loss between wild type (WT) and TRPC3/6/7-/- diabetic mice. Finally, we examined whether the TGFβ1 signaling pathway is changed in diabetic WT and TRPC3/6/7-/- mice. TRPC6 protein in the renal cortex was increased in WT diabetic mice. High glucose (HG) treatment increased TRPC6 expression in human podocytes. TRPC3 protein, however, was not altered in either diabetic mice or HG-treated human podocytes. Although diabetic WT and TRPC3/6/7-/- mice had similar levels of hyperglycemia, the TRPC3/6/7-/- diabetic mice showed less polyuria, kidney hypertrophy, glomerular enlargement, albuminuria, and had lost less podocytes compared with WT diabetic mice. In addition, we observed decreased expression of anti-apoptotic Bcl2 and increased expression of pro-apoptotic cleaved caspase 3 in WT diabetic mice, but such changes were not significant in TRPC3/6/7-/- diabetic mice. Western blot and immunohistochemistry revealed that TGFβ1, p-Smad2/3, and fibronectin were upregulated in WT diabetic mice; however, expression of these signaling molecules was not changed in TRPC3/6/7-/- diabetic mice. In conclusion, deletion of DAG-responsive TRPCs attenuates diabetic renal injury via inhibiting the upregulation of TGFβ1 signaling in diabetic kidneys.
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the contribution of trpc1 trpc3 trpc5 and trpc6 to touch and hearing
Neuroscience Letters, 2016Co-Authors: Jane E Sexton, Joel Abramowitz, Lutz Birnbaumer, Terri Desmonds, Kathryn Quick, Ruth Taylor, Andy Forge, Corne J Kros, John WoodAbstract:Transient receptor potential channels have diverse roles in mechanosensation. Evidence is accumulating that members of the canonical subfamily of TRP channels (TRPC) are involved in touch and hearing. Characteristic features of TRP channels include their high structural homology and their propensity to form heteromeric complexes which suggests potential functional redundancy. We previously showed that TRPC3 and TRPC6 double knockout animals have deficits in light touch and hearing whilst single knockouts were apparently normal. We have extended these studies to analyse deficits in global quadruple TRPC1, 3, 5 and 6 null mutant mice. We examined both touch and hearing in behavioural and electrophysiological assays, and provide evidence that the quadruple knockout mice have larger deficits than the TRPC3 TRPC6 double knockouts. Mechano-electrical transducer currents of cochlear outer hair cells were however normal. This suggests that TRPC1, TRPC3, TRPC5 and TRPC6 channels contribute to cutaneous and auditory mechanosensation in a combinatorial manner, but have no direct role in cochlear mechanotransduction.
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evidence for functional coupling of cgmp cgki signalling and trpc channels in endothelium but not in vascular smooth muscle
BMC Clinical Pharmacology, 2013Co-Authors: Florian Loga, Lutz Birnbaumer, Marc Freichel, Veit Flockerzi, Alexander Dietrich, Katrin Domes, Franz Hofmann, Jorg W WegenerAbstract:Background Signaling via cGMP-dependent protein kinase I (cGKI) is the major pathway in vascular smooth muscle (SM), by which endothelial NO regulates vascular tone. Recent evidence suggests that canonical transient receptor potential (TRPC) channels are targets of cGKI in SM and mediate the relaxant effects of cGMP signaling. We tested this concept by investigating the role of cGMP/cGKI signaling on vascular tone and peripheral resistance using Trpc6, Trpc3, Trpc3/6, Trpc1/3/6, and SM-specific cGKI (sm-cGKI) mice.
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intrinsic phototransduction persists in melanopsin expressing ganglion cells lacking diacylglycerol sensitive trpc subunits
European Journal of Neuroscience, 2011Co-Authors: Claudio E Perezleighton, Tiffany M. Schmidt, Joel Abramowitz, Lutz Birnbaumer, Paulo KofujiAbstract:In mammals, intrinsically photosensitive retinal ganglion cells (ipRGCs) mediate various non-image-forming photic responses such as circadian photoentrainment, pupillary light reflex, and pineal melatonin suppression. ipRGCs directly respond to environmental light by activation of the photopigment melanopsin followed by the opening of an unidentified cation-selective channel. Studies in heterologous expression systems and in the native retina have strongly implicated diacylglycerol-sensitive transient receptor potential channels containing TRPC3, TRPC6, and TRPC7 subunits in melanopsin-evoked depolarization. Here we show that melanopsin-evoked electrical responses largely persist in ipRGCs recorded from early postnatal (P6-P8) and adult (P22-P50) mice lacking expression of functional TRPC3, TRPC6, or TRPC7 subunits. Multielectrode array recordings performed at P6-P8 stages under conditions that prevent influences from rod/cone photoreceptors show comparable light sensitivity for the melanopsin-evoked responses in these mutant mouse lines in comparison to wild-type mice. Patch-clamp recordings from adult mouse ipRGCs lacking TRPC3 or TRPC7 subunits show intrinsic light-evoked responses equivalent to those recorded in wild-type mice. Persistence of intrinsic light-evoked responses was also noted in ipRGCs lacking TRPC6 subunits, however of significantly smaller magnitudes. These results demonstrate that the melanopsin-evoked depolarization in ipRGCs is not mediated by either TRPC3, TRPC6, or TRPC7 channel subunits alone. They also suggest that the melanopsin signaling pathway includes TRPC6-containing heteromeric channels in mature retinas.
Thomas Gudermann - One of the best experts on this subject based on the ideXlab platform.
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in vivo trpc functions in the cardiopulmonary vasculature
Cell Calcium, 2007Co-Authors: Alexander Dietrich, Hermann Kalwa, Beate Fuchs, Friedrich Grimminger, Norbert Weissmann, Thomas GudermannAbstract:Cardiovascular diseases are the leading cause of death in the industrialized countries. The cardiovascular system includes the systemic blood circulation, the heart and the pulmonary circulation providing sufficient blood flow and oxygen to peripheral tissues and organs according to their metabolic demand. This review focuses on three major cell types of the cardiovascular system: myocytes of the heart as well as smooth muscle cells and endothelial cells from the systemic and pulmonary circulation. Ion channels initiate and regulate contraction in all three cell types, and the identification of their genes has significantly improved our knowledge of signal transduction pathways in these cells. Among the ion channels expressed in smooth muscle cells, cation channels of the TRPC family allow for the entry of Na + and Ca 2+ . Physiological functions of TRPC1, TRPC3, TRPC4, TRPC5, TRPC6 and TRPC7 in the cardiovascular system, dissected by downregulating channel activity in isolated tissues or by the analysis of gene-deficient mouse models, are reviewed. Possible functional roles and physiological regulation of TRPCs as homomeric or heteromeric channels in these cell types are discussed. Moreover, TRP channels may also be responsible for pathophysiological processes of the cardiovascular system like hypertension as well as cardiac hypertrophy and increased endothelial permeability. © 2007 Elsevier Ltd. All rights reserved.
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Subunit composition of mammalian transient receptor potential channels in living cells
Proceedings of the National Academy of Sciences of the United States of America, 2002Co-Authors: Thomas Hofmann, Michael Schaefer, Günter Schultz, Thomas GudermannAbstract:Hormones, neurotransmitters, and growth factors give rise to calcium entry via receptor-activated cation channels that are activated downstream of phospholipase C activity. Members of the transient receptor potential channel (TRPC) family have been characterized as molecular substrates mediating receptor-activated cation influx. TRPC channels are assumed to be composed of multiple TRPC proteins. However, the cellular principles governing the assembly of TRPC proteins into homo- or heteromeric ion channels still remain elusive. By pursuing four independent experimental approaches—i.e., subcellular cotrafficking of TRPC subunits, differential functional suppression by dominant-negative subunits, fluorescence resonance energy transfer between labeled TRPC subunits, and coimmunoprecipitation—we investigate the combinatorial rules of TRPC assembly. Our data show that (i) TRPC2 does not interact with any known TRPC protein and (ii) TRPC1 has the ability to form channel complexes together with TRPC4 and TRPC5. (iii) All other TRPCs exclusively assemble into homo- or heterotetramers within the confines of TRPC subfamilies—e.g., TRPC4/5 or TRPC3/6/7. The principles of TRPC channel formation offer the conceptual framework to assess the physiological role of distinct TRPC proteins in living cells.
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Cloning, expression and subcellular localization of two novel splice variants of mouse transient receptor potential channel 2.
Biochemical Journal, 2000Co-Authors: Thomas Hofmann, Michael Schaefer, Günter Schultz, Thomas GudermannAbstract:Transient receptor potential channels (TRPCs) are known as candidate molecular correlates of receptor-activated or store-operated calcium entry. While functional roles for most TRPCs have been suggested, the physiological relevance of TRPC2 remains obscure. Whereas human and bovine TRPC2 are candidate pseudogenes, full-length rodent TRPC2 transcripts have been reported. There is, however, considerable controversy concerning mRNA splicing, tissue distribution and the function of these proteins. We report the molecular cloning of two novel murine TRPC2 splice variants, mTRPC2α and mTRPC2β. mTRPC2α RNA is expressed at low levels in many tissues and cell systems, while mTRPC2β is exclusively and abundantly expressed in the vomeronasal organ (VNO). When expressed in human embryonic kidney (HEK)-293 cells, mTRPC2 did not enhance receptor- or store-activated calcium entry. In order to investigate the basis of such a functional defect, mTRPC2–green fluorescent protein fusion proteins were examined by confocal microscopy. Fusion proteins were retained in endomembranes when expressed in HEK-293 or other cells of epithelial or neuronal origin. Co-expression of TRPC2 with other TRPCs did not restore plasma-membrane trafficking. We conclude that TRPC2 may form functional channels in the cellular context of the VNO, but is unlikely to have a physiological function in other tissues. The sequences of mTRPC2α and mTRPC2β have been submitted to GenBank under the accession numbers AF230802 and AF230803 respectively.
Shmuel Muallem - One of the best experts on this subject based on the ideXlab platform.
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molecular determinants mediating gating of transient receptor potential canonical trpc channels by stromal interaction molecule 1 stim1
Journal of Biological Chemistry, 2014Co-Authors: Kyu Pil Lee, Shmuel Muallem, Seok Choi, Jeong Hee Hong, Malini Ahuja, Sarabeth Graham, Dong Min Shin, Joseph P YuanAbstract:Transient receptor potential canonical (TRPC) channels mediate a critical part of the receptor-evoked Ca(2+) influx. TRPCs are gated open by the endoplasmic reticulum Ca(2+) sensor STIM1. Here we asked which stromal interaction molecule 1 (STIM1) and TRPC domains mediate the interaction between them and how this interaction is used to open the channels. We report that the STIM1 Orai1-activating region domain of STIM1 interacts with the TRPC channel coiled coil domains (CCDs) and that this interaction is essential for opening the channels by STIM1. Thus, disruption of the N-terminal (NT) CCDs by triple mutations eliminated TRPC surface localization and reduced binding of STIM1 to TRPC1 and TRPC5 while increasing binding to TRPC3 and TRPC6. Single mutations in TRPC1 NT or C-terminal (CT) CCDs reduced interaction and activation of TRPC1 by STIM1. Remarkably, single mutations in the TRPC3 NT CCD enhanced interaction and regulation by STIM1. Disruption in the TRPC3 CT CCD eliminated regulation by STIM1 and the enhanced interaction caused by NT CCD mutations. The NT CCD mutations converted TRPC3 from a TRPC1-dependent to a TRPC1-independent, STIM1-regulated channel. TRPC1 reduced the FRET between BFP-TRPC3 and TRPC3-YFP and between CFP-TRPC3-YFP upon stimulation. Accordingly, knockdown of TRPC1 made TRPC3 STIM1-independent. STIM1 dependence of TRPC3 was reconstituted by the TRPC1 CT CCD alone. Knockout of Trpc1 and Trpc3 similarly inhibited Ca(2+) influx, and inhibition of Trpc3 had no further effect on Ca(2+) influx in Trpc1(-/-) cells. Cell stimulation enhanced the formation of Trpc1-Stim1-Trpc3 complexes. These findings support a model in which the TRPC3 NT and CT CCDs interact to shield the CT CCD from interaction with STIM1. The TRPC1 CT CCD dissociates this interaction to allow the STIM1 Orai1-activating region within STIM1 access to the TRPC3 CT CCD and regulation of TRPC3 by STIM1. These studies provide evidence that the TRPC channel CCDs participate in channel gating.
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stim1 gates trpc channels but not orai1 by electrostatic interaction
Molecular Cell, 2008Co-Authors: Weizhong Zeng, Joseph P Yuan, Paul F Worley, Guo N Huang, Min Seuk Kim, Young Jin Choi, Shmuel MuallemAbstract:Summary The receptor-evoked Ca 2+ signal includes activation of the store-operated channels (SOCs) TRPCs and the Orais. Although both are gated by STIM1, it is not known how STIM1 gates the channels and whether STIM1 gates the TRPCs and Orais by the same mechanism. Here, we report the molecular mechanism by which STIM1 gates TRPC1, which involves interaction between two conserved, negatively charged aspartates in TRPC1( 639 DD 640 ) with the positively charged STIM1( 684 KK 685 ) in STIM1 polybasic domain. Charge swapping and functional analysis revealed that exact orientation of the charges on TRPC1 and STIM1 are required, but all positive-negative charge combinations on TRPC1 and STIM1, except STIM1( 684 EE 685 )+TRPC1( 639 RR 640 ), are functional as long as they are reciprocal, indicating that STIM1 gates TRPC1 by intermolecular electrostatic interaction. Similar gating was observed with TRPC3( 697 DD 698 ). STIM1 gates Orai1 by a different mechanism since the polybasic and S/P domains of STIM1 are not required for activation of Orai1 by STIM1.
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stim1 heteromultimerizes trpc channels to determine their function as store operated channels
Nature Cell Biology, 2007Co-Authors: Joseph P Yuan, Paul F Worley, Weizhong Zeng, Guo N Huang, Shmuel MuallemAbstract:Stromal interacting molecule 1 (STIM1) is a Ca2+ sensor that conveys the Ca2+ load of the endoplasmic reticulum to store-operated channels (SOCs) at the plasma membrane. Here, we report that STIM1 binds TRPC1, TRPC4 and TRPC5 and determines their function as SOCs. Inhibition of STIM1 function inhibits activation of TRPC5 by receptor stimulation, but not by La3+, suggesting that STIM1 is obligatory for activation of TRPC channels by agonists, but STIM1 is not essential for channel function. Through a distinct mechanism, STIM1 also regulates TRPC3 and TRPC6. STIM1 does not bind TRPC3 and TRPC6, and regulates their function indirectly by mediating the heteromultimerization of TRPC3 with TRPC1 and TRPC6 with TRPC4. TRPC7 is not regulated by STIM1. We propose a new definition of SOCs, as channels that are regulated by STIM1 and require the store depletion-mediated clustering of STIM1. By this definition, all TRPC channels, except TRPC7, function as SOCs.
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TRPC channels as STIM1-regulated store-operated channels.
Cell calcium, 2007Co-Authors: Paul F Worley, Joseph P Yuan, Weizhong Zeng, Guo N Huang, Joo Young Kim, Min Goo Lee, Shmuel MuallemAbstract:Receptor-activated Ca(2+) influx is mediated largely by store-operated channels (SOCs). TRPC channels mediate a significant portion of the receptor-activated Ca(2+) influx. However, whether any of the TRPC channels function as a SOC remains controversial. Our understanding of the regulation of TRPC channels and their function as SOCs is being reshaped with the discovery of the role of STIM1 in the regulation of Ca(2+) influx channels. The findings that STIM1 is an ER resident Ca(2+) binding protein that regulates SOCs allow an expanded and molecular definition of SOCs. SOCs can be considered as channels that are regulated by STIM1 and require the clustering of STIM1 in response to depletion of the ER Ca(2+) stores and its translocation towards the plasma membrane. TRPC1 and other TRPC channels fulfill these criteria. STIM1 binds to TRPC1, TRPC2, TRPC4 and TRPC5 but not to TRPC3, TRPC6 and TRPC7, and STIM1 regulates TRPC1 channel activity. Structure-function analysis reveals that the C-terminus of STIM1 contains the binding and gating function of STIM1. The ERM domain of STIM1 binds to TRPC channels and a lysine-rich region participates in the gating of SOCs and TRPC1. Knock-down of STIM1 by siRNA and prevention of its translocation to the plasma membrane inhibit the activity of native SOCs and TRPC1. These findings support the conclusion that TRPC1 is a SOC. Similar studies with other TRPC channels demonstrate their regulation by STIM1 and indicate that all TRPC channels, except TRPC7, function as SOCs.
Joseph P Yuan - One of the best experts on this subject based on the ideXlab platform.
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molecular determinants mediating gating of transient receptor potential canonical trpc channels by stromal interaction molecule 1 stim1
Journal of Biological Chemistry, 2014Co-Authors: Kyu Pil Lee, Shmuel Muallem, Seok Choi, Jeong Hee Hong, Malini Ahuja, Sarabeth Graham, Dong Min Shin, Joseph P YuanAbstract:Transient receptor potential canonical (TRPC) channels mediate a critical part of the receptor-evoked Ca(2+) influx. TRPCs are gated open by the endoplasmic reticulum Ca(2+) sensor STIM1. Here we asked which stromal interaction molecule 1 (STIM1) and TRPC domains mediate the interaction between them and how this interaction is used to open the channels. We report that the STIM1 Orai1-activating region domain of STIM1 interacts with the TRPC channel coiled coil domains (CCDs) and that this interaction is essential for opening the channels by STIM1. Thus, disruption of the N-terminal (NT) CCDs by triple mutations eliminated TRPC surface localization and reduced binding of STIM1 to TRPC1 and TRPC5 while increasing binding to TRPC3 and TRPC6. Single mutations in TRPC1 NT or C-terminal (CT) CCDs reduced interaction and activation of TRPC1 by STIM1. Remarkably, single mutations in the TRPC3 NT CCD enhanced interaction and regulation by STIM1. Disruption in the TRPC3 CT CCD eliminated regulation by STIM1 and the enhanced interaction caused by NT CCD mutations. The NT CCD mutations converted TRPC3 from a TRPC1-dependent to a TRPC1-independent, STIM1-regulated channel. TRPC1 reduced the FRET between BFP-TRPC3 and TRPC3-YFP and between CFP-TRPC3-YFP upon stimulation. Accordingly, knockdown of TRPC1 made TRPC3 STIM1-independent. STIM1 dependence of TRPC3 was reconstituted by the TRPC1 CT CCD alone. Knockout of Trpc1 and Trpc3 similarly inhibited Ca(2+) influx, and inhibition of Trpc3 had no further effect on Ca(2+) influx in Trpc1(-/-) cells. Cell stimulation enhanced the formation of Trpc1-Stim1-Trpc3 complexes. These findings support a model in which the TRPC3 NT and CT CCDs interact to shield the CT CCD from interaction with STIM1. The TRPC1 CT CCD dissociates this interaction to allow the STIM1 Orai1-activating region within STIM1 access to the TRPC3 CT CCD and regulation of TRPC3 by STIM1. These studies provide evidence that the TRPC channel CCDs participate in channel gating.
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stim1 gates trpc channels but not orai1 by electrostatic interaction
Molecular Cell, 2008Co-Authors: Weizhong Zeng, Joseph P Yuan, Paul F Worley, Guo N Huang, Min Seuk Kim, Young Jin Choi, Shmuel MuallemAbstract:Summary The receptor-evoked Ca 2+ signal includes activation of the store-operated channels (SOCs) TRPCs and the Orais. Although both are gated by STIM1, it is not known how STIM1 gates the channels and whether STIM1 gates the TRPCs and Orais by the same mechanism. Here, we report the molecular mechanism by which STIM1 gates TRPC1, which involves interaction between two conserved, negatively charged aspartates in TRPC1( 639 DD 640 ) with the positively charged STIM1( 684 KK 685 ) in STIM1 polybasic domain. Charge swapping and functional analysis revealed that exact orientation of the charges on TRPC1 and STIM1 are required, but all positive-negative charge combinations on TRPC1 and STIM1, except STIM1( 684 EE 685 )+TRPC1( 639 RR 640 ), are functional as long as they are reciprocal, indicating that STIM1 gates TRPC1 by intermolecular electrostatic interaction. Similar gating was observed with TRPC3( 697 DD 698 ). STIM1 gates Orai1 by a different mechanism since the polybasic and S/P domains of STIM1 are not required for activation of Orai1 by STIM1.
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stim1 heteromultimerizes trpc channels to determine their function as store operated channels
Nature Cell Biology, 2007Co-Authors: Joseph P Yuan, Paul F Worley, Weizhong Zeng, Guo N Huang, Shmuel MuallemAbstract:Stromal interacting molecule 1 (STIM1) is a Ca2+ sensor that conveys the Ca2+ load of the endoplasmic reticulum to store-operated channels (SOCs) at the plasma membrane. Here, we report that STIM1 binds TRPC1, TRPC4 and TRPC5 and determines their function as SOCs. Inhibition of STIM1 function inhibits activation of TRPC5 by receptor stimulation, but not by La3+, suggesting that STIM1 is obligatory for activation of TRPC channels by agonists, but STIM1 is not essential for channel function. Through a distinct mechanism, STIM1 also regulates TRPC3 and TRPC6. STIM1 does not bind TRPC3 and TRPC6, and regulates their function indirectly by mediating the heteromultimerization of TRPC3 with TRPC1 and TRPC6 with TRPC4. TRPC7 is not regulated by STIM1. We propose a new definition of SOCs, as channels that are regulated by STIM1 and require the store depletion-mediated clustering of STIM1. By this definition, all TRPC channels, except TRPC7, function as SOCs.
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TRPC channels as STIM1-regulated store-operated channels.
Cell calcium, 2007Co-Authors: Paul F Worley, Joseph P Yuan, Weizhong Zeng, Guo N Huang, Joo Young Kim, Min Goo Lee, Shmuel MuallemAbstract:Receptor-activated Ca(2+) influx is mediated largely by store-operated channels (SOCs). TRPC channels mediate a significant portion of the receptor-activated Ca(2+) influx. However, whether any of the TRPC channels function as a SOC remains controversial. Our understanding of the regulation of TRPC channels and their function as SOCs is being reshaped with the discovery of the role of STIM1 in the regulation of Ca(2+) influx channels. The findings that STIM1 is an ER resident Ca(2+) binding protein that regulates SOCs allow an expanded and molecular definition of SOCs. SOCs can be considered as channels that are regulated by STIM1 and require the clustering of STIM1 in response to depletion of the ER Ca(2+) stores and its translocation towards the plasma membrane. TRPC1 and other TRPC channels fulfill these criteria. STIM1 binds to TRPC1, TRPC2, TRPC4 and TRPC5 but not to TRPC3, TRPC6 and TRPC7, and STIM1 regulates TRPC1 channel activity. Structure-function analysis reveals that the C-terminus of STIM1 contains the binding and gating function of STIM1. The ERM domain of STIM1 binds to TRPC channels and a lysine-rich region participates in the gating of SOCs and TRPC1. Knock-down of STIM1 by siRNA and prevention of its translocation to the plasma membrane inhibit the activity of native SOCs and TRPC1. These findings support the conclusion that TRPC1 is a SOC. Similar studies with other TRPC channels demonstrate their regulation by STIM1 and indicate that all TRPC channels, except TRPC7, function as SOCs.
Michael Schaefer - One of the best experts on this subject based on the ideXlab platform.
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direct activation of trpc3 channels by the antimalarial agent artemisinin
Cells, 2020Co-Authors: Nicole Urban, Michael SchaeferAbstract:(1) Background: Members of the TRPC3/TRPC6/TRPC7 subfamily of canonical transient receptor potential (TRP) channels share an amino acid similarity of more than 80% and can form heteromeric channel complexes. They are directly gated by diacylglycerols in a protein kinase C-independent manner. To assess TRPC3 channel functions without concomitant protein kinase C activation, direct activators are highly desirable. (2) Methods: By screening 2000 bioactive compounds in a Ca2+ influx assay, we identified artemisinin as a TRPC3 activator. Validation and characterization of the hit was performed by applying fluorometric Ca2+ influx assays and electrophysiological patch-clamp experiments in heterologously or endogenously TRPC3-expressing cells. (3) Results: Artemisinin elicited Ca2+ entry through TRPC3 or heteromeric TRPC3:TRPC6 channels, but did not or only weakly activated TRPC6 and TRPC7. Electrophysiological recordings confirmed the reversible and repeatable TRPC3 activation by artemisinin that was inhibited by established TRPC3 channel blockers. Rectification properties and reversal potentials were similar to those observed after stimulation with a diacylglycerol mimic, indicating that artemisinin induces a similar active state as the physiological activator. In rat pheochromocytoma PC12 cells that endogenously express TRPC3, artemisinin induced a Ca2+ influx and TRPC3-like currents. (4) Conclusions: Our findings identify artemisinin as a new biologically active entity to activate recombinant or native TRPC3-bearing channel complexes in a membrane-confined fashion.
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Identification and Validation of Larixyl Acetate as a Potent TRPC6 Inhibitor.
Molecular pharmacology, 2015Co-Authors: Nicole Urban, Liming Wang, Wolfgang M Kuebler, Sandra Kwiek, Jörg Rademann, Michael SchaeferAbstract:Classical or canonical transient receptor potential 6 (TRPC6), a nonselective and Ca(2+)-permeable cation channel, mediates pathophysiological responses within pulmonary and renal diseases that are still poorly controlled by current medication. Thus, controlling TRPC6 activity may provide a promising and challenging pharmacological approach. Recently identified chemical entities have demonstrated that TRPC6 is pharmacologically targetable. However, isotype-selectivity with regard to its closest relative, TRPC3, is difficult to achieve. Reasoning that balsams, essential oils, or incense materials that are traditionally used for inhalation may contain biologic activities to block TRPC6 activity, we embarked on a natural compound strategy to identify new TRPC6-blocking chemical entities. Within several preparations of plant extracts, a strong TRPC6-inhibitory activity was found in conifer balsams. The biologic activity was associated with nonvolatile resins, but not with essential oils. Of various conifers, the larch balsam was unique in displaying a marked TRPC6-prevalent mode of action. By testing the main constituents of larch resin, we identified larixol and larixyl acetate as blockers of Ca(2+) entry and ionic currents through diacylglycerol- or receptor-activated recombinant TRPC6 channels, exhibiting approximately 12- and 5-fold selectivity compared with its closest relatives TRPC3 and TRPC7, respectively. No significant inhibition of more distantly related TRPV or TRPM channels was seen. The potent inhibition of recombinant TRPC6 by larixyl acetate (IC50 = 0.1-0.6 µM) was confirmed for native TRPC6-like [Ca(2+)]i signals in diacylglycerol-stimulated rat pulmonary artery smooth muscle cells. In isolated mouse lungs, larix-6-yl monoacetate (CAS 4608-49-5; larixyl acetate; 5 µM) prevented acute hypoxia-induced vasoconstriction. We conclude that larch-derived labdane-type diterpenes are TRPC6-selective inhibitors and may represent a starting point for pharmacological TRPC6 modulation within experimental therapies.
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novel pharmacological trpc inhibitors block hypoxia induced vasoconstriction
Cell Calcium, 2012Co-Authors: Nicole Urban, Kerstin Hill, Liming Wang, Wolfgang M Kuebler, Michael SchaeferAbstract:The Ca(2+)-permeable, nonselective cation channel TRPC6 is gated via phospholipase C-activating receptors and has recently been implicated in hypoxia-induced pulmonary vasoconstriction (HPV), idiopathic pulmonary hypertension and focal segmental glomerulosclerosis (FSGS). Therefore, TRPC6 is a promising target for pharmacological interference. To identify and develop TRPC6-blocking compounds, we screened the Chembionet library, a collection of 16,671 chemically diverse drug-like compounds, for biological activity to prevent the 1-oleoyl-2-acetyl-sn-glycerol-triggered Ca(2+) influx in a stably transfected HEK(TRPC6-YFP) cell line. Hits were validated and characterised by fluorometric and electrophysiological methods. Six compounds displayed inhibitory potency at low micromolar concentrations, lack of cytotoxicity and blocked the receptor-dependent mode of TRPC6 activation. The specificity was tested towards closely (TRPC3 and TRPC7) and more distantly related TRP channels. One of the compounds, 8009-5364, displayed a 2.5-fold TRPC6-selectivity compared to TRPC3, and almost no inhibition of TRPC7 or the other TRP channels tested. Block of native TRPC3/6-like responses was confirmed in dissociated pulmonary artery smooth muscle cells. Two non-polar blockers effectively suppressed the HPV responses in the perfused mouse lung model. We conclude that pharmacological targeting of TRPC6 is feasible and provide a promising concept to treat pulmonary diseases that are characterised by excessive hypoxic vasoconstriction.
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TRPC4 and TRPC5: receptor-operated Ca2+-permeable nonselective cation channels.
Cell calcium, 2003Co-Authors: Tim D Plant, Michael SchaeferAbstract:The seven mammalian channels from the classical (TRPC) subfamily of transient receptor potential (TRP) channels are thought to be receptor-operated cation channels activated in a phospholipase C (PLC)-dependent manner. Based on sequence similarity, TRPC channels can be divided into four subgroups. Group 4 comprises TRPC4 and TRPC5, and is most closely related to group 1 (TRPC1). The functional properties observed following heterologous expression of TRPC4 or TRPC5 in mammalian cells are contradictory and, therefore, controversial. In our hands, and in several independent studies, both channels, probably as homotetramers, form receptor-operated, Ca2+-permeable, nonselective cation channels activated independently of inositol 1,4,5-trisphosphate (InsP(3)) receptor activation or Ca2+ store-depletion. As heteromultimers with TRPC1, TRPC4 and TRPC5 form receptor-operated, Ca2+-permeable, nonselective cation channels with biophysical properties distinct from homomeric TRPC4 or TRPC5. In other studies, TRPC4 and TRPC5 have been shown to be store-operated channels, with moderate to high Ca2+ permeabilities. At present there is no clear explanation for these major differences in functional properties. To date, little is known as to which native cation channels are formed by TRPC4 and TRPC5. Endothelial cells from TRPC4(-/-) mice lack a highly Ca2+-permeable, store-dependent current, and data support a role for TRPC4 in endothelium-mediated vasorelaxation. A similar current in adrenal cortical cells is reduced by TRPC4 antisense. From similarities in the properties of the currents and expression of appropriate isoforms in the tissues, it is likely that heteromultimers of TRPC1 and TRPC4 or TRPC5 form receptor-operated nonselective cation channels in central neurones, and that TRPC4 contributes to nonselective cation channels in intestinal smooth muscle.
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Subunit composition of mammalian transient receptor potential channels in living cells
Proceedings of the National Academy of Sciences of the United States of America, 2002Co-Authors: Thomas Hofmann, Michael Schaefer, Günter Schultz, Thomas GudermannAbstract:Hormones, neurotransmitters, and growth factors give rise to calcium entry via receptor-activated cation channels that are activated downstream of phospholipase C activity. Members of the transient receptor potential channel (TRPC) family have been characterized as molecular substrates mediating receptor-activated cation influx. TRPC channels are assumed to be composed of multiple TRPC proteins. However, the cellular principles governing the assembly of TRPC proteins into homo- or heteromeric ion channels still remain elusive. By pursuing four independent experimental approaches—i.e., subcellular cotrafficking of TRPC subunits, differential functional suppression by dominant-negative subunits, fluorescence resonance energy transfer between labeled TRPC subunits, and coimmunoprecipitation—we investigate the combinatorial rules of TRPC assembly. Our data show that (i) TRPC2 does not interact with any known TRPC protein and (ii) TRPC1 has the ability to form channel complexes together with TRPC4 and TRPC5. (iii) All other TRPCs exclusively assemble into homo- or heterotetramers within the confines of TRPC subfamilies—e.g., TRPC4/5 or TRPC3/6/7. The principles of TRPC channel formation offer the conceptual framework to assess the physiological role of distinct TRPC proteins in living cells.