Michael Schaefer

Extracellular Ca2+ is a danger signal activating the NLRP3 inflammasome through G protein-coupled calcium sensing receptors.

Activation of the NLRP3 inflammasome enables monocytes and macrophages to release high levels of interleukin-1β during inflammatory responses. Concentrations of extracellular calcium can increase at sites of infection, inflammation or cell activation. Here we show that increased extracellular calcium activates the NLRP3 inflammasome via stimulation of G protein-coupled calcium sensing receptors. Activation is mediated by signalling through the calcium-sensing receptor and GPRC6A via the phosphatidyl inositol/Ca2+ pathway. The resulting increase in the intracellular calcium concentration triggers inflammasome assembly and Caspase-1 activation. We identified necrotic cells as one source for excess extracellular calcium triggering this activation. In vivo, increased calcium concentrations can amplify the inflammatory response in the mouse model of carrageenan-induced footpad swelling, and this effect was inhibited in GPRC6A−/− mice. Our results demonstrate that G-protein-coupled receptors can activate the inflammasome, and indicate that increased extracellular calcium has a role as a danger signal and amplifier of inflammation.

Positive allosteric modulation by ivermectin of human but not murine P2X7 receptors.

In mammalian cells, the anti‐parasitic drug ivermectin is known as a positive allosteric modulator of the ATP‐activated ion channel P2X4 and is used to discriminate between P2X4‐ and P2X7‐mediated cellular responses. In this paper we provide evidence that the reported isoform selectivity of ivermectin is a species‐specific phenomenon.

Novel pharmacological TRPC inhibitors block hypoxia-induced vasoconstriction.

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.

A small molecule restores function to TRPML1 mutant isoforms responsible for mucolipidosis type IV

Mucolipidosis type IV (MLIV) is an autosomal recessive lysosomal storage disorder often characterized by severe neurodevelopmental abnormalities and neuro-retinal degeneration. Mutations in the TRPML1 gene are causative for MLIV. We used lead optimization strategies to identify--and MLIV patient fibroblasts to test--small-molecule activators for their potential to restore TRPML1 mutant channel function. Using the whole-lysosome planar patch-clamp technique, we found that activation of MLIV mutant isoforms by the endogenous ligand PI(3,5)P2 is strongly reduced, while activity can be increased using synthetic ligands. We also found that the F465L mutation renders TRPML1 pH insensitive, while F408Δ impacts synthetic ligand binding. Trafficking defects and accumulation of zinc in lysosomes of MLIV mutant fibroblasts can be rescued by the small molecule treatment. Collectively, our data demonstrate that small molecules can be used to restore channel function and rescue disease associated abnormalities in patient cells expressing specific MLIV point mutations.

Cross-inhibition between native and recombinant TRPV1 and P2X3 receptors

ABSTRACT Small‐ to medium‐sized neurons in the dorsal root ganglion (DRG) convey nociceptive information to the spinal cord. The co‐expression of TRPV1 receptors (sensitive to vanilloids, heat and acidic pH) with P2X3 receptors (sensitive to extracellular ATP) has been found in many DRG neurons. We investigated whether the co‐activation of these two receptor classes in small‐diameter cells leads to a modulation of the resulting current responses shaping the intensity of pain sensation. The whole‐cell patch clamp method was used to record agonist‐induced currents in cultured rat DRG neurons and in HEK293 cells transfected with the respective wild‐type recombinant receptors or their mutants. Co‐immunoprecipitation studies were used to demonstrate the physical association of TRPV1 and P2X3 receptors. At a negative holding potential, the P2X3 receptor agonist α,β‐meATP induced less current in the presence of the TRPV1 agonist capsaicin than that in its absence. This inhibitory interaction was not changed at a positive holding potential, in a Ba2+‐containing superfusion medium, or when the buffering of intrapipette Ca2+ was altered. The C‐terminal truncation at Glu362 of P2X3 receptors abolished the TRPV1/P2X3 cross‐talk in the HEK293 expression system. Co‐immunoprecipitation studies with polyclonal antibodies generated against TRPV1 and P2X3 showed a visible signal in HEK293 cells transfected with both receptors. It is concluded that the two pain‐relevant receptors TRPV1 and P2X3 interact with each other in an inhibitory manner probably by a physical association established by a motif located at the C‐terminal end of the P2X3 receptor distal to Glu362.

Heterodimerization of Hypothalamic G-Protein-Coupled Receptors Involved in Weight Regulation

Background: Melanocortin 3 and 4 receptors (MC3R and MC4R) are known to play an essential role in hypothalamic weight regulation. In addition to these two G-protein-coupled receptors (GPCRs), a huge number of other GPCRs are expressed in hypothalamic regions, and some of them are involved in weight regulation. So far, homodimerization was shown for a few of these receptors. Heterodimerization of unrelated receptors may have profound functional consequence but heterodimerization of GPCRs involved in weight regulation was not reported yet. Methods: A selective number of hypothalamically expressed GPCRs were cloned into a eukaryotic expression vector. Cell surface expression was demonstrated by an ELISA approach. Subcellular distribution was investigated by confocal laser microscopy. A sandwich ELISA and fluorescence resonance energy transfer (FRET) were used to determine protein-protein interaction. Results: Via sandwich ELISA and FRET approach we could demonstrate a robust interaction of the MC4R with GPR7, both of which are expressed in the hypothalamic nucleus paraventricularis. Moreover, we determined a strong interaction of MC3R with the growth hormone secretagogue receptor expressed in the nucleus arcuatus. Conclusion: Identification GPCR heterodimerization adds to the understanding of the complexity of weight regulation and may provide important information to develop therapeutic strategies to treat obesity.

Novel APP/Aβ mutation K16N produces highly toxic heteromeric Aβ oligomers

Here, we describe a novel missense mutation in the amyloid precursor protein (APP) causing a lysine‐to‐asparagine substitution at position 687 (APP770; herein, referred to as K16N according to amyloid‐β (Aβ) numbering) resulting in an early onset dementia with an autosomal dominant inheritance pattern. The K16N mutation is located exactly at the α‐secretase cleavage site and influences both APP and Aβ. First, due to the K16N mutation APP secretion is affected and a higher amount of Aβ peptides is being produced. Second, Aβ peptides carrying the K16N mutation are unique in that the peptide itself is not harmful to neuronal cells. Severe toxicity, however, is evident upon equimolar mixture of wt and mutant peptides, mimicking the heterozygous state of the subject. Furthermore, Aβ42 K16N inhibits fibril formation of Aβ42 wild‐type. Even more, Aβ42 K16N peptides are protected against clearance activity by the major Aβ‐degrading enzyme neprilysin. Thus the mutation characterized here harbours a combination of risk factors that synergistically may contribute to the development of early onset Alzheimer disease.

P2X7 receptors at adult neural progenitor cells of the mouse subventricular zone

Neurogenesis requires the balance between the proliferation of newly formed progenitor cells and subsequent death of surplus cells. RT-PCR and immunocytochemistry demonstrated the presence of P2X7 receptor mRNA and immunoreactivity in cultured neural progenitor cells (NPCs) prepared from the adult mouse subventricular zone (SVZ). Whole-cell patch-clamp recordings showed a marked potentiation of the inward current responses both to ATP and the prototypic P2X7 receptor agonist dibenzoyl-ATP (Bz-ATP) at low Ca(2+) and zero Mg(2+) concentrations in the bath medium. The Bz-ATP-induced currents reversed their polarity near 0 mV; in NPCs prepared from P2X7(-/-) mice, Bz-ATP failed to elicit membrane currents. The general P2X/P2Y receptor antagonist PPADS and the P2X7 selective antagonists Brilliant Blue G and A-438079 strongly depressed the effect of Bz-ATP. Long-lasting application of Bz-ATP induced an initial current, which slowly increased to a steady-state response. In combination with the determination of YO-PRO uptake, these experiments suggest the dilation of a receptor-channel and/or the recruitment of a dye-uptake pathway. Ca(2+)-imaging by means of Fura-2 revealed that in a Mg(2+)-deficient bath medium Bz-ATP causes [Ca(2+)](i) transients fully depending on the presence of external Ca(2+). The MTT test indicated a concentration-dependent decrease in cell viability by Bz-ATP treatment. Correspondingly, Bz-ATP led to an increase in active caspase 3 immunoreactivity, indicating a P2X7-controlled apoptosis. In acute SVZ brain slices of transgenic Tg(nestin/EGFP) mice, patch-clamp recordings identified P2X7 receptors at NPCs with pharmacological properties identical to those of their cultured counterparts. We suggest that the apoptotic/necrotic P2X7 receptors at NPCs may be of particular relevance during pathological conditions which lead to increased ATP release and thus could counterbalance the ensuing excessive cell proliferation.

Citrus fruit and fabacea secondary metabolites potently and selectively block TRPM3.

The melastatin‐related transient receptor potential TRPM3 is a calcium‐permeable nonselective cation channel that can be activated by the neurosteroid pregnenolone sulphate (PregS) and heat. TRPM3‐deficient mice show an impaired perception of noxious heat. Hence, drugs inhibiting TRPM3 possibly get in focus of analgesic therapy.

Clemizole Hydrochloride Is a Novel and Potent Inhibitor of Transient Receptor Potential Channel TRPC5

Canonical transient receptor potential channel 5 (TRPC5) is a nonselective, Ca2+-permeable cation channel that belongs to the large family of transient receptor potential channels. It is predominantly found in the central nervous system with a high expression density in the hippocampus, the amygdala, and the frontal cortex. Several studies confirm that TRPC5 channels are implicated in the regulation of neurite length and growth cone morphology. We identified clemizole as a novel inhibitor of TRPC5 channels. Clemizole efficiently blocks TRPC5 currents and Ca2+ entry in the low micromolar range (IC50 = 1.0–1.3 µM), as determined by fluorometric intracellular free Ca2+ concentration ([Ca2+]i) measurements and patch-clamp recordings. Clemizole blocks TRPC5 currents irrespectively of the mode of activation, for example, stimulation of G protein–coupled receptors, hypo-osmotic buffer conditions, or by the direct activator riluzole. Electrophysiological whole-cell recordings revealed that the block was mostly reversible. Moreover, clemizole was still effective in blocking TRPC5 single channels in excised inside-out membrane patches, hinting to a direct block of TRPC5 by clemizole. Based on fluorometric [Ca2+]i measurements, clemizole exhibits a sixfold selectivity for TRPC5 over TRPC4β (IC50 = 6.4 µM), the closest structural relative of TRPC5, and an almost 10-fold selectivity over TRPC3 (IC50 = 9.1 µM) and TRPC6 (IC50 = 11.3 µM). TRPM3 and M8 as well as TRPV1, V2, V3, and V4 channels were only weakly affected by markedly higher clemizole concentrations. Clemizole was not only effective in blocking heterologously expressed TRPC5 homomers but also TRPC1:TRPC5 heteromers as well as native TRPC5-like currents in the U-87 glioblastoma cell line.