Michaela Lichtenegger

Novel pyrazole compounds for pharmacological discrimination between receptor-operated and store-operated Ca2+ entry pathways

Pyrazole derivatives have recently been suggested as selective blockers of transient receptor potential cation (TRPC) channels but their ability to distinguish between the TRPC and Orai pore complexes is ill‐defined. This study was designed to characterize a series of pyrazole derivatives in terms of TRPC/Orai selectivity and to delineate consequences of selective suppression of these pathways for mast cell activation.

PKC-dependent coupling of calcium permeation through transient receptor potential canonical 3 (TRPC3) to calcineurin signaling in HL-1 myocytes

Cardiac transient receptor potential canonical (TRPC) channels are crucial upstream components of Ca2+/calcineurin/nuclear factor of activated T cells (NFAT) signaling, thereby controlling cardiac transcriptional programs. The linkage between TRPC-mediated Ca2+ signals and NFAT activity is still incompletely understood. TRPC conductances may govern calcineurin activity and NFAT translocation by supplying Ca2+ either directly through the TRPC pore into a regulatory microdomain or indirectly via promotion of voltage-dependent Ca2+ entry. Here, we show that a point mutation in the TRPC3 selectivity filter (E630Q), which disrupts Ca2+ permeability but preserves monovalent permeation, abrogates agonist-induced NFAT signaling in HEK293 cells as well as in murine HL-1 atrial myocytes. The E630Q mutation fully retains the ability to convert phospholipase C-linked stimuli into L-type (CaV1.2) channel-mediated Ca2+ entry in HL-1 cells, thereby generating a dihydropyridine-sensitive Ca2+ signal that is isolated from the NFAT pathway. Prevention of PKC-dependent modulation of TRPC3 by either inhibition of cellular kinase activity or mutation of a critical phosphorylation site in TRPC3 (T573A), which disrupts targeting of calcineurin into the channel complex, converts cardiac TRPC3-mediated Ca2+ signaling into a transcriptionally silent mode. Thus, we demonstrate a dichotomy of TRPC-mediated Ca2+ signaling in the heart constituting two distinct pathways that are differentially linked to gene transcription. Coupling of TRPC3 activity to NFAT translocation requires microdomain Ca2+ signaling by PKC-modified TRPC3 complexes. Our results identify TRPC3 as a pivotal signaling gateway in Ca2+-dependent control of cardiac gene expression.

TRPC3 contributes to regulation of cardiac contractility and arrhythmogenesis by dynamic interaction with NCX1

Aim TRPC3 is a non-selective cation channel, which forms a Ca2+ entry pathway involved in cardiac remodelling. Our aim was to analyse acute electrophysiological and contractile consequences of TRPC3 activation in the heart. Methods and results We used a murine model of cardiac TRPC3 overexpression and a novel TRPC3 agonist, GSK1702934A, to uncover (patho)physiological functions of TRPC3. GSK1702934A induced a transient, non-selective conductance and prolonged action potentials in TRPC3-overexpressing myocytes but lacked significant electrophysiological effects in wild-type myocytes. GSK1702934A transiently enhanced contractility and evoked arrhythmias in isolated Langendorff hearts from TRPC3-overexpressing but not wild-type mice. Interestingly, pro-arrhythmic effects outlasted TRPC3 current activation, were prevented by enhanced intracellular Ca2+ buffering, and suppressed by the NCX inhibitor 3′,4′-dichlorobenzamil hydrochloride. GSK1702934A substantially promoted NCX currents in TRPC3-overexpressing myocytes. The TRPC3-dependent electrophysiologic, pro-arrhythmic, and inotropic actions of GSK1702934A were mimicked by angiotensin II (AngII). Immunocytochemistry demonstrated colocalization of TRPC3 with NCX1 and disruption of local interaction upon channel activation by either GSK1702934A or AngII. Conclusion Cardiac TRPC3 mediates Ca2+ and Na+ entry in proximity of NCX1, thereby elevating cellular Ca2+ levels and contractility. Excessive activation of TRPC3 is associated with transient cellular Ca2+ overload, spatial uncoupling between TRPC3 and NCX1, and arrhythmogenesis. We propose TRPC3-NCX micro/nanodomain communication as determinant of cardiac contractility and susceptibility to arrhythmogenic stimuli.

TRPC3: a multifunctional signaling molecule.

TRPC3 represents one of the first identified mammalian relative of the Drosophila trp gene product. Despite extensive biochemical and biophysical characterization as well as ambitious attempts to uncover its physiological role in native cell systems, the channel protein still represents a rather enigmatic member of the TRP superfamily. TRPC3 is significantly expressed in the brain and heart and appears of (patho)physiological importance in both non-excitable and excitable cells, being potentially involved in a wide spectrum of Ca(2+) signaling mechanisms. TRPC3 cation channels display unique gating and regulatory properties that allow for recognition and integration of multiple input stimuli including lipid mediators, cellular Ca(2+) gradients, as well as redox signals. Physiological/pathophysiological functions of this highly versatile cation channel protein are as yet incompletely understood. Its ability to associate in a dynamic manner with a variety of partner proteins enables TRPC3 to serve coordination of multiple downstream signaling pathways and control of divergent cellular functions. Here, we summarize current knowledge on ion channel features as well as possible signaling functions of TRPC3 and discuss the potential biological relevance of this signaling molecule.

A novel homology model of TRPC3 reveals allosteric coupling between gate and selectivity filter

Utilizing a novel molecular model of TRPC3, based on the voltage-gated sodium channel from Arcobacter butzleri (Na(V)AB) as template, we performed structure-guided mutagenesis experiments to identify amino acid residues involved in divalent permeation and gating. Substituted cysteine accessibility screening within the predicted selectivity filter uncovered amino acids 629-631 as the narrowest part of the permeation pathway with an estimated pore diameter of < 5.8Å. E630 was found to govern not only divalent permeability but also sensitivity of the channel to block by ruthenium red. Mutations in a hydrophobic cluster at the cytosolic termini of transmembrane segment 6, corresponding to the S6 bundle crossing structure in Na(V)AB, distorted channel gating. Removal of a large hydrophobic residue (I667A or I667E) generated channels with approximately 60% constitutive activity, suggesting I667 as part of the dynamic structure occluding the permeation path. Destabilization of the gate was associated with reduced Ca2+ permeability, altered cysteine cross-linking in the selectivity filter and promoted channel block by ruthenium red. Collectively, we present a structural model of the TRPC3 permeation pathway and localize the channels selectivity filter and the occluding gate. Moreover, we provide evidence for allosteric coupling between the gate and the selectivity filter in TRPC3.

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

AbstractTransient 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.A photoswitchable diacylglycerol enabled a screen that found critical TRPC3 lipid-sensing residues and identified a lateral fenestration in the pore domain that allows lipids to protrude toward the permeation pathway to control channel 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.

Analysis of the Molecular Basis of Ca2+- dependent Regulation of TRPC3 Channels

TRPC3 generates non-selective cation channels that are subject to a highly complex regulation by the permeating cation Ca2+. This Ca2+-mediated control of TRPC3 is considered to include mechanisms of negative feedback regulation (current inactivation) essential for physiological roles of the channel complex. Ca2+ ions may control TRPC channel function by interaction with regulatory proteins that are located in, or targeted to a cytoplasmic regulatory microdomain or alternatively by certain interactions with the permeation pathway. To explore the molecular basis of TRPC3 modulation by Ca2+, we tested the impact of Ca2+ permeation and of negative feedback regulation by PKC on Ca2+ sensitivity of TRPC3 channels expressed in HEK293 cells. TRPC3 currents were rapidly suppressed by elevation of extracellular Ca2+ (0.8 to 2 mM) range after activation via muscarinic receptor stimulation in nominally Ca2+-free solution, and removal of divalents from the extracellular solution activated a TRPC3-mediated conductance. Neutralization of a single negative charge within the putative pore domain (E630Q) generated channels which were largely insensitive to changes in extracellular divalents but displayed normal current decay during activating stimuli. Mutation in T573, which is required for down-regulation of channels by PKC-phosphorylation (T573A; “moonwalker”) was associated with a strongly reduced current decay. We conclude that TRPC3 channels sense the extracellular Ca2+ concentration by a Ca2+ binding site within the permeation pathway, while current decay after stimulation is barely dependent on Ca2+ permeation through the channel but involves regulatory phosphorylation.Supported by FWF, P21925-B19.

Molecular engineering of the TRPC3 pore structure identifies Ca2+ permeation through TRPC3 channels as a key determinant of cardiac calcineurin/NFAT signaling.

Results Elimination of Ca permeation through TRPC3 abrogated its ability to trigger NFAT translocation in both HEK293 cells and in HL-1 atrial myocytes. Wild-type TRPC3 was found capable of initiating NFAT translocation in atrial myocytes by a small, homogenous elevation of cytoplasmic Ca that was independent of voltagegated CaV1.2 channels. By contrast, a Ca 2+ impermeant TRPC3 mutant strongly promoted endothelin-induced Ca signals in HL1 cells via enhanced activity of CaV1.2 channels without concomitant NFAT translocation. Conclusions Our results demonstrate two strictly separated Ca signaling functions of cardiac TRPC3 channels as well as a tight and efficient link between TRPC3-mediated Ca permeation and calcineurin/NFAT signaling.