Analysis of hyperforin (St. John's wort) action at TRPC6 channel leads to the development of a new class of antidepressant drugs.

St. John's wort is an herb, long used in folk medicine for the treatment of mild depression. Its antidepressant constituent, hyperforin, has properties such as chemical instability and induction of drug-drug interactions that preclude its use for individual pharmacotherapies. Here we identify the transient receptor potential canonical 6 channel (TRPC6) as a druggable target to control anxious and depressive behavior and as a requirement for hyperforin antidepressant action. We demonstrate that TRPC6 deficiency in mice not only results in anxious and depressive behavior, but also reduces excitability of hippocampal CA1 pyramidal neurons and dentate gyrus granule cells. Using electrophysiology and targeted mutagenesis, we show that hyperforin activates the channel via a specific binding motif at TRPC6. We performed an analysis of hyperforin action to develop a new antidepressant drug that uses the same TRPC6 target mechanism for its antidepressant action. We synthesized the hyperforin analog Hyp13, which shows similar binding to TRPC6 and recapitulates TRPC6-dependent anxiolytic and antidepressant effects in mice. Hyp13 does not activate pregnan-X-receptor (PXR) and thereby loses the potential to induce drug-drug interactions. This may provide a new approach to develop better treatments for depression, since depression remains one of the most treatment-resistant mental disorders, warranting the development of effective drugs based on naturally occurring compounds.

Computer-Based Drug Design of Positive Modulators of Store-Operated Calcium Channels to Prevent Synaptic Dysfunction in Alzheimer's Disease.

Store-operated calcium entry (SOCE) constitutes a fine-tuning mechanism responsible for the replenishment of intracellular stores. Hippocampal SOCE is regulated by store-operated channels (SOC) organized in tripartite complex TRPC6/ORAI2/STIM2. It is suggested that in neurons, SOCE maintains intracellular homeostatic Ca2+ concentration at resting conditions and is needed to support the structure of dendritic spines. Recent evidence suggests that positive modulators of SOC are prospective drug candidates to treat Alzheimer's disease (AD) at early stages. Although STIM2 and ORAI2 are definitely involved in the regulation of nSOC amplitude and a play major role in AD pathogenesis, growing evidence suggest that it is not easy to target these proteins pharmacologically. Existing positive modulators of TRPC6 are unsuitable for drug development due to either bad pharmacokinetics or side effects. Thus, we concentrate the review on perspectives to develop specific nSOC modulators based on available 3D structures of TRPC6, ORAI2, and STIM2. We shortly describe the structural features of existing models and the methods used to prepare them. We provide commonly used steps applied for drug design based on 3D structures of target proteins that might be used to develop novel AD preventing therapy.

GSK1702934A and M085 directly activate TRPC6 via a mechanism of stimulating the extracellular cavity formed by the pore helix and transmembrane helix S6.

Transient receptor potential canonical (TRPC) channels, as important membrane proteins regulating intracellular calcium (Ca2+i) signaling, are involved in a variety of physiological and pathological processes. Activation and regulation of TRPC are more dependent on membrane or intracellular signals. However, how extracellular signals regulate TRPC6 function remains to be further investigated. Here, we suggest that two distinct small molecules, M085 and GSK1702934A, directly activate TRPC6, both through a mechanism of stimulation of extracellular sites formed by the pore helix (PH) and transmembrane (TM) helix S6. In silico docking scanning of TRPC6 identified three extracellular sites that can bind small molecules, of which only mutations on residues of PH and S6 helix significantly reduced the apparent affinity of M085 and GSK1702934A and attenuated the maximal response of TRPC6 to these two chemicals by altering channel gating of TRPC6. Combing metadynamics, molecular dynamics simulations, and mutagenesis, we revealed that W679, E671, E672, and K675 in the PH and N701 and Y704 in the S6 helix constitute an orthosteric site for the recognition of these two agonists. The importance of this site was further confirmed by covalent modification of amino acid residing at the interface of the PH and S6 helix. Given that three structurally distinct agonists M085, GSK1702934A, and AM-0883, act at this site, as well as the occupancy of lipid molecules at this position found in other TRP subfamilies, it is suggested that the cavity formed by the PH and S6 has an important role in the regulation of TRP channel function by extracellular signals.

Novel Mechanistic Insights and Potential Therapeutic Impact of TRPC6 in Neurovascular Coupling and Ischemic Stroke.

Ischemic stroke is one of the most disabling diseases and a leading cause of death globally. Despite advances in medical care, the global burden of stroke continues to grow, as no effective treatments to limit or reverse ischemic injury to the brain are available. However, recent preclinical findings have revealed the potential role of transient receptor potential cation 6 (TRPC6) channels as endogenous protectors of neuronal tissue. Activating TRPC6 in various cerebral ischemia models has been found to prevent neuronal death, whereas blocking TRPC6 enhances sensitivity to ischemia. Evidence has shown that Ca2+ influx through TRPC6 activates the cAMP (adenosine 3',5'-cyclic monophosphate) response element-binding protein (CREB), an important transcription factor linked to neuronal survival. Additionally, TRPC6 activation may counter excitotoxic damage resulting from glutamate release by attenuating the activity of N-methyl-d-aspartate (NMDA) receptors of neurons by posttranslational means. Unresolved though, are the roles of TRPC6 channels in non-neuronal cells, such as astrocytes and endothelial cells. Moreover, TRPC6 channels may have detrimental effects on the blood-brain barrier, although their exact role in neurovascular coupling requires further investigation. This review discusses evidence-based cell-specific aspects of TRPC6 in the brain to assess the potential targets for ischemic stroke management.

Structural basis for pharmacological modulation of the TRPC6 channel.

Transient receptor potential canonical (TRPC) proteins form nonselective cation channels that play physiological roles in a wide variety of cells. Despite growing evidence supporting the therapeutic potential of TRPC6 inhibition in treating pathological cardiac and renal conditions, mechanistic understanding of TRPC6 function and modulation remains obscure. Here we report cryo-EM structures of TRPC6 in both antagonist-bound and agonist-bound states. The structures reveal two novel recognition sites for the small-molecule modulators corroborated by mutagenesis data. The antagonist binds to a cytoplasm-facing pocket formed by S1-S4 and the TRP helix, whereas the agonist wedges at the subunit interface between S6 and the pore helix. Conformational changes upon ligand binding illuminate a mechanistic rationale for understanding TRPC6 modulation. Furthermore, structural and mutagenesis analyses suggest several disease-related mutations enhance channel activity by disrupting interfacial interactions. Our results provide principles of drug action that may facilitate future design of small molecules to ameliorate TRPC6-mediated diseases.

Mammalian TRP ion channels are insensitive to membrane stretch.

TRP channels of the transient receptor potential ion channel superfamily are involved in a wide variety of mechanosensory processes, including touch sensation, pain, blood pressure regulation, bone loading and detection of cerebrospinal fluid flow. However, in many instances it is unclear whether TRP channels are the primary transducers of mechanical force in these processes. In this study, we tested stretch activation of eleven TRP channels from six mammalian subfamilies. We found that these TRP channels were insensitive to short membrane stretches in cellular systems. Furthermore, we purified TRPC6 and demonstrated its insensitivity to stretch in liposomes, an artificial bilayer system free from cellular components. Additionally, we demonstrated that, when expressed in C. elegans neurons, mouse TRPC6 restores the mechanoresponse of a touch insensitive mutant but requires diacylglycerol for activation. These results strongly suggest that the mammalian members of the TRP ion channel family are insensitive to tension induced by cell membrane stretching and, thus, are more likely to be activated by cytoplasmic tethers or downstream components and to act as amplifiers of cellular mechanosensory signaling cascades.

Small Fluorescein Arsenical Hairpin-Based Förster Resonance Energy Transfer Analysis Reveals Changes in Amino- to Carboxyl-Terminal Interactions upon OAG Activation of Classical Transient Receptor Potential 6.

Although the overall structure of many classical transient receptor potential proteins (TRPC), including human and murine TRPC6, were recently resolved by cryoelectron microscopy analysis, structural changes during channel activation by 1-oleoyl-1-acetyl-sn-glycerol (OAG), the membrane-permeable analog of diacylglycerol, were not defined. Moreover, data on carboxyl- and amino-terminal interactions were not provided, as the amino-terminal regions of murine and human TRPC6 were not resolved. Therefore, we employed a Förster resonance energy transfer (FRET) approach using a small fluorescein arsenical hairpin (FlAsH) targeted to a short tetracysteine sequence at the unresolved amino-terminus and cerulean, a cyan fluorescent protein, as a tag at the carboxyl-terminus of the murine TRPC6 protein. After OAG as well as GSK-1702934A activation, FRET efficiency was simultaneously and significantly reduced, indicating a decreased interaction between the amino to carboxyl termini in the functional tagged murine TRPC6 tetramer (TRPC6 WT) heterologously expressed in human embryonic kidney 293T cells. There was a significant reduction in the FRET signal obtained from analysis of murine TRPC6 FRET constructs with homologous amino-terminal mutations (M131T, G108S) that had been identified in human patients with inherited focal segmental glomerulosclerosis, a condition that can lead to end-stage renal disease. A novel, designed loss-of-function TRPC6 mutation (N109A) in the amino-terminus in close proximity to the carboxyl-terminus produced similar FRET ratios. SIGNIFICANCE STATEMENT: Our data show for the first time that FlAsH-tagging of ion channels is a promising tool to study conformational changes after channel opening and may significantly advance the analysis of ion channel activation as well as their mutants involved in channelopathies.

Soluble klotho regulates TRPC6 calcium signaling via lipid rafts, independent of the FGFR-FGF23 pathway.

Soluble klotho (sKlotho), the shed ectodomain of α-klotho, protects the heart by down-regulating transient receptor potential canonical isoform 6 (TRPC6)-mediated calcium signaling. Binding to α2-3-sialyllactose moiety of gangliosides in lipid rafts and inhibition of raft-dependent signaling underlies the mechanism. A recent 3-Å X-ray structure of sKlotho in complex with fibroblast growth factor receptor (FGFR) and fibroblast growth factor 23 (FGF23) indicates that its ß6α6 loop might block access to the proposed binding site for α2-3-sialyllactose. It was concluded that sKlotho only functions in complex with FGFR and FGF23 and that sKlotho's pleiotropic effects all depend on FGF23. Here, we report that sKlotho can inhibit TRPC6 channels expressed in cells lacking endogenous FGFRs. Structural modeling and molecular docking show that a repositioned ß6α6 loop allows sKlotho to bind α2-3-sialyllactose. Molecular dynamic simulations further show the α2-3-sialyllactose-bound sKlotho complex to be stable. Domains mimicking sKlotho's sialic acid-recognizing activity inhibit TRPC6. The results strongly support the hypothesis that sKlotho can exert effects independent of FGF23 and FGFR.-Wright, J. D., An, S.-W., Xie, J., Lim, C., Huang, C.-L. Soluble klotho regulates TRPC6 calcium signaling via lipid rafts, independent of the FGFR-FGF23 pathway.

Cryo-EM structure of the cytoplasmic domain of murine transient receptor potential cation channel subfamily C member 6 (TRPC6).

The kidney maintains the internal milieu by regulating the retention and excretion of proteins, ions, and small molecules. The glomerular podocyte forms the slit diaphragm of the ultrafiltration filter, whose damage leads to progressive kidney failure and focal segmental glomerulosclerosis (FSGS). The canonical transient receptor potential 6 (TRPC6) ion channel is expressed in the podocyte, and mutations in its cytoplasmic domain cause FSGS in humans. In vitro evaluation of disease-causing mutations in TRPC6 has revealed that these genetic alterations result in abnormal ion channel gating. However, the mechanism whereby the cytoplasmic domain modulates TRPC6 function is largely unknown. Here, we report a cryo-EM structure of the cytoplasmic domain of murine TRPC6 at 3.8 Å resolution. The cytoplasmic fold of TRPC6 is characterized by an inverted dome-like chamber pierced by four radial horizontal helices that converge into a vertical coiled-coil at the central axis. Unlike other TRP channels, TRPC6 displays a unique domain swap that occurs at the junction of the horizontal helices and coiled-coil. Multiple FSGS mutations converge at the buried interface between the vertical coiled-coil and the ankyrin repeats, which form the dome, suggesting these regions are critical for allosteric gating modulation. This functionally critical interface is a potential target for drug design. Importantly, dysfunction in other family members leads to learning deficits (TRPC1/4/5) and ataxia (TRPC3). Our data provide a structural framework for the mechanistic investigation of the TRPC family.

Structure of the receptor-activated human TRPC6 and TRPC3 ion channels.

TRPC6 and TRPC3 are receptor-activated nonselective cation channels that belong to the family of canonical transient receptor potential (TRPC) channels. They are activated by diacylglycerol, a lipid second messenger. TRPC6 and TRPC3 are involved in many physiological processes and implicated in human genetic diseases. Here we present the structure of human TRPC6 homotetramer in complex with a newly identified high-affinity inhibitor BTDM solved by single-particle cryo-electron microscopy to 3.8 Å resolution. We also present the structure of human TRPC3 at 4.4 Å resolution. These structures show two-layer architectures in which the bell-shaped cytosolic layer holds the transmembrane layer. Extensive inter-subunit interactions of cytosolic domains, including the N-terminal ankyrin repeats and the C-terminal coiled-coil, contribute to the tetramer assembly. The high-affinity inhibitor BTDM wedges between the S5-S6 pore domain and voltage sensor-like domain to inhibit channel opening. Our structures uncover the molecular architecture of TRPC channels and provide a structural basis for understanding the mechanism of these channels.

Proline-dependent and basophilic kinases phosphorylate human TRPC6 at serine 14 to control channel activity through increased membrane expression.

Signaling via the transient receptor potential (TRP) ion channel C6 plays a pivotal role in hereditary and sporadic glomerular kidney disease. Several studies have identified gain-of-function mutations of TRPC6 and report induced expression and enhanced channel activity of TRPC6 in association with glomerular diseases. Interfering with TRPC6 activity may open novel therapeutic pathways. TRPC6 channel activity is controlled by protein expression and stability as well as intracellular trafficking. Identification of regulatory phosphorylation sites in TRPC6 and corresponding protein kinases is essential to understand the regulation of TRPC6 activity and may result in future therapeutic strategies. In this study, an unbiased phosphoproteomic screen of human TRPC6 identified several novel serine phosphorylation sites. The phosphorylation site at serine 14 of TRPC6 is embedded in a basophilic kinase motif that is highly conserved across species. We confirmed serine 14 as a target of MAPKs and proline-directed kinases like cyclin-dependent kinase 5 (Cdk5) in cell-based as well as in vitro kinase assays and quantitative phosphoproteomic analysis of TRPC6. Phosphorylation of TRPC6 at serine 14 enhances channel conductance by boosting membrane expression of TRPC6, whereas protein stability and multimerization of TRPC6 are not altered, making serine 14 phosphorylation a potential drug target to interfere with TRPC6 channel activity.-Hagmann, H., Mangold, N., Rinschen, M. M., Koenig, T., Kunzelmann, K., Schermer, B., Benzing, T., Brinkkoetter, P. T. Proline-dependent and basophilic kinases phosphorylate human TRPC6 at serine 14 to control channel activity through increased membrane expression.