The Knockout for G Protein-Coupled Receptor-Like PfSR25 Increases the Susceptibility of Malaria Parasites to the Antimalarials Lumefantrine and Piperaquine but Not to Medicine for Malaria Venture Compounds

Previously we have reported that the G protein-coupled receptor (GPCR)-like PfSR25 in Plasmodium falciparum is a potassium (K+) sensor linked to intracellular calcium signaling and that knockout parasites (PfSR25-) are more susceptible to oxidative stress and antimalarial compounds. Here, we explore the potential role of PfSR25 in susceptibility to the antimalarial compounds atovaquone, chloroquine, dihydroartemisinin, lumefantrine, mefloquine, piperaquine, primaquine, and pyrimethamine and the Medicine for Malaria Venture (MMV) compounds previously described to act on egress/invasion (MMV006429, MMV396715, MMV019127, MMV665874, MMV665878, MMV665785, and MMV66583) through comparative assays with PfSR25- and 3D7 parasite strains, using flow cytometry assays. The IC50 and IC90 results show that lumefantrine and piperaquine have greater activity on the PfSR25- parasite strain when compared to 3D7. For MMV compounds, we found no differences between the strains except for the compound MMV665831, which we used to investigate the store-operated calcium entry (SOCE) mechanism. The results suggest that PfSR25 may be involved in the mechanism of action of the antimalarials lumefantrine and piperaquine. Our data clearly show that MMV665831 does not affect calcium entry in parasites after we depleted their internal calcium pools with thapsigargin. The results demonstrated here shed light on new possibilities on the antimalarial mechanism, bringing evidence of the involvement of the GPCR-like PfSR25.

Calcium-ligand variants of the myocilin olfactomedin propeller selected from invertebrate phyla reveal cross-talk with N-terminal blade and surface helices

Olfactomedins are a family of modular proteins found in multicellular organisms that all contain five-bladed β-propeller olfactomedin (OLF) domains. In support of differential functions for the OLF propeller, the available crystal structures reveal that only some OLF domains harbor an internal calcium-binding site with ligands derived from a triad of residues. For the myocilin OLF domain (myoc-OLF), ablation of the ion-binding site (triad Asp, Asn, Asp) by altering the coordinating residues affects the stability and overall structure, in one case leading to misfolding and glaucoma. Bioinformatics analysis reveals a variety of triads with possible ion-binding characteristics lurking in OLF domains in invertebrate chordates such as Arthropoda (Asp–Glu–Ser), Nematoda (Asp–Asp–His) and Echinodermata (Asp–Glu–Lys). To test ion binding and to extend the observed connection between ion binding and distal structural rearrangements, consensus triads from these phyla were installed in the myoc-OLF. All three protein variants exhibit wild-type-like or better stability, but their calcium-binding properties differ, concomitant with new structural deviations from wild-type myoc-OLF. Taken together, the results indicate that calcium binding is not intrinsically destabilizing to myoc-OLF or required to observe a well ordered side helix, and that ion binding is a differential feature that may underlie the largely elusive biological function of OLF propellers.

Astrocytes integrate and drive neural activity

SUMMARYMany brain functions depend on the ability of neural networks to temporally integrate transient inputs to produce sustained discharges. This can occur through cell-autonomous mechanisms in individual neurons or through reverberating activity in recurrently connected neural networks. We report a third mechanism involving temporal integration of neural activity by a network of astrocytes. Previously, we showed that some types of interneurons can generate long-lasting trains of action potentials for tens of seconds (“barrage firing”) following repeated depolarizing stimuli. Here, we show that calcium signaling in an astrocytic network correlates with barrage firing; that active depolarization of astrocyte networks by chemical or optogenetic stimulation enhances barrage firing; and that chelating internal calcium, inhibiting release from internal stores, or inhibiting GABA transporters or metabotropic glutamate receptors inhibited barrage firing. Thus, through complex molecular processes, networks of interconnected astrocytes influence the spatiotemporal dynamics of neural networks by directly integrating neural activity and driving long-lasting barrages of action potentials in some populations of inhibitory interneurons.

Ergosterol induces mobilization of internal calcium in tobacco cells

As for natural sterols, only ergosterol is recognized very specifically and sensitively (nM) by plants cells. Ergosterol interacts with tobacco suspension cells and trigger pH changes of extracellular medium, oxidative burst and synthesis of phytoalexins. Compared with the responses induced by cryptogein, a proteinaceous elicitor from Phytophthora sp., oxidative burst, DpH and phytoalexin accumulation were weaker with ergosterol. Cryptogein stimulated an apparent continuous uptake of external calcium within 40 min, whereas no net uptake of external calcium occurred upon the addition of ergosterol. However, the elicitation with either cryptogein or ergosterol resulted in an increase of the fluorescence of calcium green 1 in cytosol. The use of several inhibitors of calcium channels (La³⁺, TMB-8, verapamil, ruthenium red, nifedipine) and a protein-kinase inhibitors (staurosporin, NPC-15437, H-89) suggests that the elicitation with ergosterol includes the mobilization of internal calcium stores in vacuoles mediated by IP3 and some protein kinases.

Mechanisms Contributing to Tonic Release at the Cone Photoreceptor Ribbon Synapse

Time-resolved capacitance measurements in combination with fluorescence measurements of internal calcium suggested three kinetic components of release in acutely isolated cone photoreceptors of the tiger salamander. A 45-fF releasable pool, corresponding to about 1,000 vesicles, was identified. This pool could be depleted with a time constant of a few hundred milliseconds and its recovery from depletion was quite rapid (τ ≈ 1 s). The fusion of vesicles in this pool was blocked by low-millimolar EGTA. Endocytosis was sufficiently slow that it is likely that refilling of the releasable pool occurred from preformed vesicles. A second, slower component of release (τdepletion ≈ 3 s) was identified that was approximately twice the size of the releasable pool. This pool may serve as a first reserve pool that replenishes the releasable pool. Computer simulations indicate that the properties of the releasable and first reserve pools are sufficient to maintain synaptic signaling for several seconds in the face of near-maximal stimulations and in the absence of other sources of vesicles. Along with lower rates of depletion, additional mechanisms, such as replenishment from distal reserve pools and the fast recycling of vesicles, may further contribute to the maintenance of graded, tonic release from cone photoreceptors.

Potassium channel blockers quinidine and caesium halt cell proliferation in C6 glioma cells via a polyamine-dependent mechanism

Potassium channels are ubiquitous in cells and serve essential functions in physiology and pathophysiology. Potassium channel blockers have been shown to block tumour growth by arresting cells at the G0/G1 checkpoint of the cell cycle. We investigated the effect of quinidine and caesium (Cs+) on cell proliferation, LDH (lactate dehydrogenase) release, free internal calcium, membrane potential, polyamine concentration, ODC (ornithine decarboxylase) activity and polyamine uptake in C6 glioma cells. The EC50 for reducing cell proliferation was 112 μM for quinidine, whereas Cs+ was less effective with an EC50 of 4.75 mM. KCl or sucrose did not affect proliferation. LDH release was augmented by quinidine. Quinidine caused a transient increase in free internal calcium but decreased calcium after a 48 h incubation period. Further 300 μM quinidine depolarized the cell membrane in a similar range as did 30 mM KCl. Quinidine decreased cellular putrescine beyond detection levels while spermidine and spermine remained unaffected. ODC activity was reduced. Addition of putrescine could not override the antiproliferative effect owing to a reduced activity of the polyamine transporter. Our study indicates that the antiproliferative effect of quinidine is not due to a simple membrane depolarization but is caused by a block of ODC activity.