An optically controlled probe identifies lipid-gating fenestrations within the TRPC3 channel

Lichtenegger, Michaela; Tiapko, Oleksandra; Svobodová, Barbora; Stockner, Thomas; Glasnov, Toma N.; Schultze-Seemann, Wolfgang; Platzer, Dieter; Cruz, Gema Guedes de la; Krenn, Sarah; Schober, Romana; Shrestha, Niroj; Schindl, Rainer; Romanin, Christoph; Gröschner, Klaus

doi:10.1038/s41589-018-0015-6

Cited by 91 publications

(152 citation statements)

References 32 publications

(31 reference statements)

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“…In addition, mutation of W680 to alanine in hTRPC6, corresponding to W577A in hTRPC5, abolished OAG activation of hTRPC6 ( 44 ). Mutations of G709/G640 in hTRPC6/3, corresponding to G606 in hTRPC5 in IBP-B, blunted PLC-mediated activation of TRPC6/3, altered responses to DAGs of TRPC3, and also altered the pattern of photoactivation of TRPC6/3 by a photo-switchable DAG analogue, OptoDArG ( 47 ). These data collectively suggest this lipid binding pocket is important for the function of TRPC channels and ligands binding at IBP-B might affect TRPC channel gating.…”

Section: Discussionmentioning

confidence: 99%

Structural basis for human TRPC5 channel inhibition by two distinct inhibitors

Song

Wei

Guo

et al. 2020

Preprint

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17TRPC5 channel is a non-selective cation channel that participates diverse physiological 18 processes. Human TRPC5 inhibitors show promise in the treatment of anxiety disorder, 19 depression and kidney disease. Despite the high relevance of TRPC5 to human health, its 20 inhibitor binding pockets have not been fully characterized due to the lack of structural 21 information, which greatly hinders structure-based drug discovery. Here we show cryo-EM 22 structures of human TRPC5 in complex with two distinct inhibitors, namely clemizole and 23 HC-070, to the resolution of 2.7 Å. Based on the high-quality cryo-EM maps, we uncover the 24 different binding pockets and detailed binding modes for these two inhibitors. Clemizole 25 binds inside the voltage sensor-like domain of each subunit, while HC-070 binds close to the 26 ion channel pore and is wedged between adjacent subunits. Both of them exert the inhibitory 27 function by stabilizing the ion channel in a closed state. These structures provide templates 28 for further design and optimization of inhibitors targeting human TRPC5. 29 70 inhibitory mechanism of these two distinct inhibitors against human TRPC5 (hTRPC5), we 71 embarked structural studies of hTRPC5 in complex with CMZ or HC-070. Our high 72 resolution cryo-EM maps are sufficient to identify their binding pockets and will aid further 73 drug development. 74 75 76 Results 77 78 Structure of human TRPC5 in complex with inhibitors 79To reveal the binding sites of CMZ and HC-070 on hTRPC5, we expressed hTRPC5 in 80 HEK293F cells for structural studies. Similar to the mouse TRPC5 counterpart (mTRPC5) 81 (33), C-terminal truncation of hTRPC5 generated a construct (hTRPC5 1-764 ) that showed 82 higher expression level of the tetramer in comparison with the full length wild type hTRPC5 83 ( Fig. S1A). Moreover, we found hTRPC5 1-764 retained the pharmacological properties of full 84 length hTRPC5 such as activation by EA ( Fig. S1, B to E). Therefore, we used hTRPC5 1-764 85 protein for structural studies. Tetrameric hTRPC5 1-764 channel was purified in detergent 86 micelles (Fig. S1F and G). To obtain the CMZ-bound and the HC-070-bound TRPC5 87 structures, CMZ or HC-070 was added to the concentrated hTRPC5 1-764 protein for cryo-EM 88 sample preparation. We obtained maps of hTRPC5 in CMZ-bound state and HC-070-bound 89 state to the resolution of 2.7 Å (Fig. 1, Figs. S2 to S5; table S1). The map qualities were 90 sufficient for model building and ligand assignment (Fig. S3, D and E, and Fig. S5, D and E).91The overall architecture of inhibitor-bound hTRPC5 is similar to the structure of mTRPC5 in 92 apo state (33), occupying 100 Å × 100 Å × 130 Å in three-dimensional space (Fig. 1). The 93 four-fold symmetric channel has two layer architecture, composed of the intracellular 94 cytosolic domain (ICD) layer and the transmembrane domain (TMD) layer (Fig. 1). In ICD 95 layer, the N-terminal ankyrin repeats domain (ARD) is below the linker-helix domain (LHD) 96 region (Fig .1F). The TRP helix mediates interactions...

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Section: Discussionmentioning

confidence: 99%

Structural basis for human TRPC5 channel inhibition by two distinct inhibitors

Song

Wei

Guo

et al. 2020

Preprint

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“…Here we provide evidence for coupling between the activity of recombinant TRPC3 channels and NFAT1 localization by utilizing precise control of channel activity by TRPC photopharmacology and chemogenetics. Direct, light-mediated control over activation and deactivation of TRPC3 wild-type and mutant channels (G652A), which displays supersensitivity to benzimidazoles and reduced basal activity [17,18], revealed a close relation between constitutive TRPC3 activity and NFAT1 signaling. This concept was supported by the observation of constitutive NFAT1 activity exclusively in cells overexpressing WT but not in cells overexpressing G652A mutant.…”

Section: Discussionmentioning

confidence: 99%

Light-Mediated Control over TRPC3-Mediated NFAT Signaling

Graziani¹,

Bacsa²,

Krivić³

et al. 2020

Cells

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Canonical transient receptor potential (TRPC) channels were identified as key players in maladaptive remodeling, with nuclear factor of activated T-cells (NFAT) transcription factors serving as downstream targets of TRPC-triggered Ca2+ entry in these pathological processes. Strikingly, the reconstitution of TRPC-NFAT signaling by heterologous expression yielded controversial results. Specifically, nuclear translocation of NFAT1 was found barely responsive to recombinant TRPC3, presumably based on the requirement of certain spatiotemporal signaling features. Here, we report efficient control of NFAT1 nuclear translocation in human embryonic kidney 293 (HEK293) cells by light, using a new photochromic TRPC benzimidazole activator (OptoBI-1) and a TRPC3 mutant with modified activator sensitivity. NFAT1 nuclear translocation was measured along with an all-optical protocol to record local and global Ca2+ pattern generated during light-mediated activation/deactivation cycling of TRPC3. Our results unveil the ability of wild-type TRPC3 to produce constitutive NFAT nuclear translocation. Moreover, we demonstrate that TRPC3 mutant that lacks basal activity enables spatiotemporally precise control over NFAT1 activity by photopharmacology. Our results suggest tight linkage between TRPC3 activity and NFAT1 nuclear translocation based on global cellular Ca2+ signals.

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“…Even the structure of TRPC3 could not represent DAG-binding pockets despite long-history for DAG-mediated gating mechanism of the channel [55,56]. One study, however, proposed a promising result [91]. Using a modeled structure of TRPC3 with TRPV1 as a template, putative DAG binding domain (G652) was postulated.…”

Section: Structure-function Relationship In G-protein Mediated Gatingmentioning

confidence: 99%

Structure–Function Relationship and Physiological Roles of Transient Receptor Potential Canonical (TRPC) 4 and 5 Channels

Kim

Hong

et al. 2019

Cells

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The study of the structure–function relationship of ion channels has been one of the most challenging goals in contemporary physiology. Revelation of the three-dimensional (3D) structure of ion channels has facilitated our understanding of many of the submolecular mechanisms inside ion channels, such as selective permeability, voltage dependency, agonist binding, and inter-subunit multimerization. Identifying the structure–function relationship of the ion channels is clinically important as well since only such knowledge can imbue potential therapeutics with practical possibilities. In a sense, recent advances in the understanding of the structure–relationship of transient receptor potential canonical (TRPC) channels look promising since human TRPC channels are calcium-permeable, non-selective cation channels expressed in many tissues such as the gastrointestinal (GI) tract, kidney, heart, vasculature, and brain. TRPC channels are known to regulate GI contractility and motility, pulmonary hypertension, right ventricular hypertrophy, podocyte injury, seizure, fear, anxiety-like behavior, and many others. In this article, we tried to elaborate recent findings of Cryo-EM (cryogenic-electron microscopy) based structural information of TRPC 4 and 5 channels and domain-specific functions of the channel, such as G-protein mediated activation mechanism, extracellular modification of the channel, homo/hetero-tetramerization, and pharmacological gating mechanisms.

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An optically controlled probe identifies lipid-gating fenestrations within the TRPC3 channel

Cited by 91 publications

References 32 publications

Structural basis for human TRPC5 channel inhibition by two distinct inhibitors

Structural basis for human TRPC5 channel inhibition by two distinct inhibitors

Light-Mediated Control over TRPC3-Mediated NFAT Signaling

Structure–Function Relationship and Physiological Roles of Transient Receptor Potential Canonical (TRPC) 4 and 5 Channels

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