PIP2 modulates TRPC3 activity via TRP helix and S4-S5 linker.

The transient receptor potential canonical type 3 (TRPC3) channel plays a pivotal role in regulating neuronal excitability in the brain via its constitutive activity. The channel is intricately regulated by lipids and has previously been demonstrated to be positively modulated by PIP2. Using molecular dynamics simulations and patch clamp techniques, we reveal that PIP2 predominantly interacts with TRPC3 at the L3 lipid binding site, located at the intersection of pre-S1 and S1 helices. We demonstrate that PIP2 sensing involves a multistep mechanism that propagates from L3 to the pore domain via a salt bridge between the TRP helix and S4-S5 linker. Notably, we find that both stimulated and constitutive TRPC3 activity require PIP2. These structural insights into the function of TRPC3 are invaluable for understanding the role of the TRPC subfamily in health and disease, in particular for cardiovascular diseases, in which TRPC3 channels play a major role.

Calcium transport and sensing in TRPC channels - New insights into a complex feedback regulation.

Canonical TRP (TRPC) channels are a still enigmatic family of signaling molecules with multimodal sensing features. These channels enable Ca2+ influx through the plasma membrane to control a diverse range of cellular functions. Based on both regulatory- and recently uncovered structural features, TRPC channels are considered to coordinate Ca2+ and other divalent cations not only within the permeation path but also at additional sensory sites. Analysis of TRPC structures by cryo-EM identified multiple regulatory ion binding pockets. With this review, we aim at an overview and a critical discussion of the current concepts of divalent sensing by TRPC channels.

TRPC3 governs the spatiotemporal organization of cellular Ca2+ signatures by functional coupling to IP3 receptors.

Communication between TRPC channels and IP3 receptors (IP3R) is considered pivotal in the generation of spatiotemporal Ca2+signaling patterns. Here we revisited the role of TRPC3-IP3R coupling for local Ca2+ signaling within TRPC3-harbouring micro/nanodomains using R-GECO as a reporter, fused to the channel´s C-terminus. Cytoplasmic Ca2+ changes at TRPC3 originated from both the entry of Ca2+ through the TRPC channel and Ca2+ mobilization via IP3R. Local Ca2+ changes at TRPC3 channels expressed in HEK293 cells were predominantly biphasic with IP3R-dependent initial Ca2+ transients, while exclusively monophasic signals were recorded when all three IP3R isoforms were lacking. Abrogation of Ca2+ entry through TRPC3 by point mutations, which impair Ca2+ permeability (E630Q), cation permeation (E630K), or DAG sensitivity (G652A), promoted microdomain Ca2+ oscillations. Ca2+ signals at E630Q, E630K, and G652A channels featured initial Ca2+ transients along with oscillatory activity. Similarly, when extracellular Ca2+ was omitted, IP3R-mediated Ca2+ transients and Ca2+ oscillations were promoted at the cytoplasmic face of wild-type TRPC3 channels. By contrast, oscillations, as well as initial Ca2+ transients, were virtually lacking, when the TRPC3 channels were sensitized by preexposure to low-level PLC activity. TIRF imaging provided evidence for dynamic colocalization of TRPC3 and IP3R. We suggest that TRPC3-mediated Ca2+ entry controls IP3R activity at ER-PM junctions to determine Ca2+ signaling signatures and enable specificity of downstream signaling.

Characterization of DAG Binding to TRPC Channels by Target-Dependent cis-trans Isomerization of OptoDArG.

Azobenzene-based photochromic lipids are valuable probes for the analysis of ion channel-lipid interactions. Rapid photoisomerization of these molecules enables the analysis of lipid gating kinetics and provides information on lipid sensing. Thermal relaxation of the metastable cis conformation to the trans conformation of azobenzene photolipids is rather slow in the dark and may be modified by ligand-protein interactions. Cis photolipid-induced changes in pure lipid membranes as visualized from the morphological response of giant unilamellar vesicles indicated that thermal cis-trans isomerization of both PhoDAG-1 and OptoDArG is essentially slow in the lipid bilayer environment. While the currents activated by cis PhoDAG remained stable upon termination of UV light exposure (dark, UV-OFF), cis OptoDArG-induced TRPC3/6/7 activity displayed a striking isoform-dependent exponential decay. The deactivation kinetics of cis OptoDArG-induced currents in the dark was sensitive to mutations in the L2 lipid coordination site of TRPC channels. We conclude that the binding of cis OptoDArG to TRPC channels promotes transition of cis OptoDArG to the trans conformation. This process is suggested to provide valuable information on DAG-ion channel interactions and may enable highly selective photopharmacological interventions.

Diacylglycerols interact with the L2 lipidation site in TRPC3 to induce a sensitized channel state.

Coordination of lipids within transient receptor potential canonical channels (TRPCs) is essential for their Ca2+ signaling function. Single particle cryo-EM studies identified two lipid interaction sites, designated L1 and L2, which are proposed to accommodate diacylglycerols (DAGs). To explore the role of L1 and L2 in TRPC3 function, we combined structure-guided mutagenesis and electrophysiological recording with molecular dynamics (MD) simulations. MD simulations indicate rapid DAG accumulation within both L1 and L2 upon its availability within the plasma membrane. Electrophysiological experiments using a photoswitchable DAG-probe reveal potentiation of TRPC3 currents during repetitive activation by DAG. Importantly, initial DAG exposure generates a subsequently sensitized channel state that is associated with significantly faster activation kinetics. TRPC3 sensitization is specifically promoted by mutations within L2, with G652A exhibiting sensitization at very low levels of active DAG. We demonstrate the ability of TRPC3 to adopt a closed state conformation that features partial lipidation of L2 sites by DAG and enables fast activation of the channel by the phospholipase C-DAG pathway.

Light Stimulation of Neurons on Organic Photocapacitors Induces Action Potentials with Millisecond Precision.

Nongenetic optical control of neurons is a powerful technique to study and manipulate the function of the nervous system. This research has benchmarked the performance of organic electrolytic photocapacitor (OEPC) optoelectronic stimulators at the level of single mammalian cells: human embryonic kidney (HEK) cells with heterologously expressed voltage-gated K+ channels and hippocampal primary neurons. OEPCs act as extracellular stimulation electrodes driven by deep red light. The electrophysiological recordings show that millisecond light stimulation of OEPC shifts conductance-voltage plots of voltage-gated K+ channels by ≈30 mV. Models are described both for understanding the experimental findings at the level of K+ channel kinetics in HEK cells, as well as elucidating interpretation of membrane electrophysiology obtained during stimulation with an electrically floating extracellular photoelectrode. A time-dependent increase in voltage-gated channel conductivity in response to OEPC stimulation is demonstrated. These findings are then carried on to cultured primary hippocampal neurons. It is found that millisecond time-scale optical stimuli trigger repetitive action potentials in these neurons. The findings demonstrate that OEPC devices enable the manipulation of neuronal signaling activities with millisecond precision. OEPCs can therefore be integrated into novel in vitro electrophysiology protocols, and the findings can inspire in vivo applications.

TRPC3, an underestimated, universal pacemaker channel?

Transient receptor potential channel canonical 3 (TRPC3) is a cation channel with poor Ca2+ selectivity and significant constitutive activity. One of the channels' features is its striking ability to couple in a surprisingly versatile manner to different down-stream signaling pathways, thereby serving cellular functions in a tissue specific manner. Expression of this protein is prominent in excitable cells, and its activity has repeatedly been implicated in electrical pacemaking. Previous studies demonstrated a linkage between constitutive activity of TRPC3 and neuronal firing in hippocampus and cerebellum. A most recent report from the Park laboratory corroborates the concept of TRPC3 functioning as a critical element in the neuronal pacemaking machinery for dopaminergic neurons of substantia nigra. Conclusively, mechanistic coupling between TRPC3 activity and firing frequency appears evident for different types of neurons, highlighting the potential of TRPC3 as a universal as well as multifunctional pacemaker channel.

Mechanisms and significance of Ca2+ entry through TRPC channels.

The transient receptor potential (TRP) superfamily of plasma membrane cation channels has been recognized as a signaling hub in highly diverse settings of human physiopathology. In the past three decades of TRP research, attention was focused mainly on the channels Ca2+ signaling function, albeit additional cellular functions, aside of providing a Ca2+ entry pathway, have been identified. Our understanding of Ca2+ signaling by TRP proteins has recently been advanced by a gain in high-resolution structure information on these pore complexes, and by the development of novel tools to investigate their role in spatiotemporal Ca2+ handling. This review summarizes recent discoveries as well as remaining, unresolved aspects of the canonical subfamily of transient receptor potential channels (TRPC) research. We aim at a concise overview on current mechanistic concepts of Ca2+ entry through- and Ca2+ signaling by TRPC channels.

Light-Mediated Control over TRPC3-Mediated NFAT Signaling.

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.

Pharmaco-Optogenetic Targeting of TRPC Activity Allows for Precise Control Over Mast Cell NFAT Signaling.

Canonical transient receptor potential (TRPC) channels are considered as elements of the immune cell Ca2+ handling machinery. We therefore hypothesized that TRPC photopharmacology may enable uniquely specific modulation of immune responses. Utilizing a recently established TRPC3/6/7 selective, photochromic benzimidazole agonist OptoBI-1, we set out to test this concept for mast cell NFAT signaling. RBL-2H3 mast cells were found to express TRPC3 and TRPC7 mRNA but lacked appreciable Ca2+/NFAT signaling in response to OptoBI-1 photocycling. Genetic modification of the cells by introduction of single recombinant TRPC isoforms revealed that exclusively TRPC6 expression generated OptoBI-1 sensitivity suitable for opto-chemical control of NFAT1 activity. Expression of any of three benzimidazole-sensitive TRPC isoforms (TRPC3/6/7) reconstituted plasma membrane TRPC conductances in RBL cells, and expression of TRPC6 or TRPC7 enabled light-mediated generation of temporally defined Ca2+ signaling patterns. Nonetheless, only cells overexpressing TRPC6 retained essentially low basal levels of NFAT activity and displayed rapid and efficient NFAT nuclear translocation upon OptoBI-1 photocycling. Hence, genetic modification of the mast cells' TRPC expression pattern by the introduction of TRPC6 enables highly specific opto-chemical control over Ca2+ transcription coupling in these immune cells.

FKBP52 regulates TRPC3-dependent Ca2+ signals and the hypertrophic growth of cardiomyocyte cultures.

The transient receptor potential (TRP; C-classical, TRPC) channel TRPC3 allows a cation (Na+/Ca2+) influx that is favored by the stimulation of Gq protein-coupled receptors (GPCRs). An enhanced TRPC3 activity is related to adverse effects, including pathological hypertrophy in chronic cardiac disease states. In the present study, we identified FK506-binding protein 52 (FKBP52, also known as FKBP4) as a novel interaction partner of TRPC3 in the heart. FKBP52 was recovered from a cardiac cDNA library by a C-terminal TRPC3 fragment (amino acids 742-848) in a yeast two-hybrid screen. Downregulation of FKBP52 promoted a TRPC3-dependent hypertrophic response in neonatal rat cardiomyocytes (NRCs). A similar effect was achieved by overexpressing peptidyl-prolyl isomerase (PPIase)-deficient FKBP52 mutants. Mechanistically, expression of the FKBP52 truncation mutants elevated TRPC3-mediated currents and Ca2+ fluxes, and the activation of calcineurin and the nuclear factor of activated T-cells in NRCs. Our data demonstrate that FKBP52 associates with TRPC3 via an as-yet-undescribed binding site in the C-terminus of TRPC3 and modulates TRPC3-dependent Ca2+ signals in a PPIase-dependent manner. This functional interaction might be crucial for limiting TRPC3-dependent signaling during chronic hypertrophic stimulation.

Photopharmacology and opto-chemogenetics of TRPC channels-some therapeutic visions.

Non-selective cation conductances formed by transient receptor potential canonical (TRPC) proteins govern the function and fate of a wide range of human cell types. In the past decade, evidence has accumulated for a pivotal role of these channels in human diseases, raising substantial interest in their therapeutic targeting. As yet, an appreciable number of small molecules for block and modulation of recombinant TRPC conductances have been identified. However, groundbreaking progress in TRPC pharmacology towards therapeutic applications is lagging behind due to incomplete understanding of their molecular pharmacology and their exact role in disease. A major breakthrough that is expected to overcome these hurdles is the recent success in obtaining high-resolution structure information on TRPC channel complexes and the advent of TRP photopharmacology and optogenetics. Here, we summarize current concepts of enhancing the precision of therapeutic interference with TRPC signaling and TRPC-mediated pathological processes.

Lipid-independent control of endothelial and neuronal TRPC3 channels by light.

Lipid-gated TRPC channels are highly expressed in cardiovascular and neuronal tissues. Exerting precise pharmacological control over their activity in native cells is expected to serve as a basis for the development of novel therapies. Here we report on a new photopharmacological tool that enables manipulation of TRPC3 channels by light, in a manner independent of lipid metabolism and with higher temporal precision than lipid photopharmacology. Using the azobenzene photoswitch moiety, we modified GSK1702934A to generate light-controlled TRPC agonists. We obtained one light-sensitive molecule (OptoBI-1) that allows us to exert efficient, light-mediated control over TRPC3 activity and the associated cellular Ca2+ signaling. OptoBI-1 enabled high-precision, temporal control of TRPC3-linked cell functions such as neuronal firing and endothelial Ca2+ transients. With these findings, we introduce a novel photopharmacological strategy to control native TRPC conductances.

TRPC3 as a Target of Novel Therapeutic Interventions.

TRPC3 is one of the classical members of the mammalian transient receptor potential (TRP) superfamily of ion channels. TRPC3 is a molecule with intriguing sensory features including the direct recognition of and activation by diacylglycerols (DAG). Although TRPC3 channels are ubiquitously expressed, they appear to control functions of the cardiovascular system and the brain in a highly specific manner. Moreover, a role of TRPC3 in immunity, cancer, and tissue remodeling has been proposed, generating much interest in TRPC3 as a target for pharmacological intervention. Advances in the understanding of molecular architecture and structure-function relations of TRPC3 have been the foundations for novel therapeutic approaches, such as photopharmacology and optochemical genetics of TRPC3. This review provides an account of advances in therapeutic targeting of TRPC3 channels.

Revelation of an enigmatic signaling machinery-First insights into the mammalian TRPC architecture.

Canonical TRP channels (TRPCs) are a particularly enigmatic family of signaling molecules with multimodal sensing features, being involved in a wide range of biological functions. Until very recently, the main hurdle towards comprehensive mechanistic understanding of TRPC signaling has been the lack of structural information. This has changed early this year by several reports on TRPC architectures resolved by single particle cryo-EM analysis. These studies confirmed recently elaborated concepts on TRPC structure-function relations, and unveiled unanticipated features and complexity in the TRPC sensing machinery.

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

Transient receptor potential canonical (TRPC) channels TRPC3, TRPC6 and TRPC7 are able to sense the lipid messenger diacylglycerol (DAG). The DAG-sensing and lipid-gating processes in these ion channels are still unknown. To gain insights into the lipid-sensing principle, we generated a DAG photoswitch, OptoDArG, that enabled efficient control of TRPC3 by light. A structure-guided mutagenesis screen of the TRPC3 pore domain unveiled a single glycine residue behind the selectivity filter (G652) that is exposed to lipid through a subunit-joining fenestration. Exchange of G652 with larger residues altered the ability of TRPC3 to discriminate between different DAG molecules. Light-controlled activation-deactivation cycling of TRPC3 channels by an OptoDArG-mediated optical 'lipid clamp' identified pore domain fenestrations as pivotal elements of the channel´s lipid-sensing machinery. We provide evidence for a novel concept of lipid sensing by TRPC channels based on a lateral fenestration in the pore domain that accommodates lipid mediators to control gating.

Intensified Microwave-Assisted N-Acylation Procedure - Synthesis and Activity Evaluation of TRPC3 Channel Agonists with a 1,3-Dihydro-2H-benzo[d]imidazol-2-one Core.

Upon controlled microwave heating and using cyanuric chloride as a coupling reagent, an efficient amidation procedure for the synthesis of 1,3-dihydro-2H-benzo[d]imidazol-2-one-based agonists of TRPC3/6 ion channels has been developed. Compared to the few conventional protocols, a drastic reduction in processing time from ca. 2 days down to 10 minutes was achieved accompanied by significantly improved product yields. The robustness of the method was confirmed by 18 additional examples including aromatic, aliphatic, and heterocyclic amines and acids. The obtained agonists were screened for biological activity at 1 µM concentration and few structure-activity relations have been established.

Optopharmacological control of TRPC channels by coumarin-caged lipids is associated with a phototoxic membrane effect.

Photouncaging of second messengers has been successfully employed to gain mechanistic insight of cellular signaling pathways. One of the most enigmatic processes of ion channel regulation is lipid recognition and lipid-gating of TRPC channels, which represents pivotal mechanisms of cellular Ca(2+) homeostasis. Recently, optopharmacological tools including caged lipid mediators became available, enabling an unprecedented level of temporal and spatial control of the activating lipid species within a cellular environment. Here we tested a commonly used caged ligand approach for suitability to investigate TRPC signaling at the level of membrane conductance and cellular Ca(2+) handling. We report a specific photouncaging artifact that is triggered by the cage structure coumarin at UV illumination. Electrophysiological characterization identified a light-dependent membrane effect of coumarin. UV light (340 nm) as used for photouncaging, initiated a membrane conductance specifically in the presence of coumarin as low as 30 µmol L(-1) concentrations. This conductance masked the TRPC3 conductance evoked by photouncaging, while TRPC-mediated cellular Ca(2+) responses were largely preserved. The observed light-induced membrane effects of the released caging moiety may well interfere with certain cellular functions, and prompt caution in using coumarin-caged second messengers in cellular studies.