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Cholera is a global health problem with no targeted therapies. The Ca2+-sensing receptor (CaSR) is a regulator of intestinal ion transport and a therapeutic target for diarrhea, and Ca2+ is considered its main agonist. We found that increasing extracellular Ca2+ had a minimal effect on forskolin-induced Cl- secretion in human intestinal epithelial T84 cells. However, extracellular Mg2+, an often-neglected CaSR agonist, suppressed forskolin-induced Cl- secretion in T84 cells by 65% at physiological levels seen in stool (10 mM). The effect of Mg2+ occurred via the CaSR/Gq signaling that led to cAMP hydrolysis. Mg2+ (10 mM) also suppressed Cl- secretion induced by cholera toxin, heat-stable E. coli enterotoxin, and vasoactive intestinal peptide by 50%. In mouse intestinal closed loops, luminal Mg2+ treatment (20 mM) inhibited cholera toxin-induced fluid accumulation by 40%. In a mouse intestinal perfusion model of cholera, addition of 10 mM Mg2+ to the perfusate reversed net fluid transport from secretion to absorption. These results suggest that Mg2+ is the key CaSR activator in mouse and human intestinal epithelia at physiological levels in stool. Since stool Mg2+ concentrations in patients with cholera are essentially zero, oral Mg2+ supplementation, alone or in an oral rehydration solution, could be a potential therapy for cholera and other cyclic nucleotide-mediated secretory diarrheas.

Some γ-glutamyl peptides including glutathione (γ-Glu-Cys-Gly) and γ-glutamyl-valyl-glycine (γ-Glu-Val-Gly= γ-EVG) are reported to increase the intensity of basic tastes, such as salty, sweet, and umami, although they have no taste themselves at tested concentrations. The mechanism of action of γ-glutamyl peptides is not clearly understood, but the calcium sensing receptor (CaSR) may be involved. Glutathione and γ-EVG enhance the pungency of some spices, and the present study investigated the effects of γ-EVG on the responses of trigeminal ganglion (TG) cells to thermosensitiveTRP channel agonists. Single-cell RT-PCR revealed that most CaSR-expressing cells co-expressed TRPV1 (sensitive to capsaicin) and TRPA1 (sensitive to allyl isothiocyanate= AITC). Intracellular Ca2+ imaging showed that pretreatment with γ-EVG excited 7% of trigeminal ganglion (TG) cells and increased the amplitude of their responses to AITC, but not to capsaicin or menthol. The enhancing effect of γ-EVG was prevented by a CaSR inhibitor. The results indicate that γ-EVG increases AITC pungency by activating a subset of trigeminal ganglion cells that co-express CaSR and TRPA1.

Midget and parasol ganglion cells (GCs) represent the major output channels from the primate eye to the brain. On-type midget and parasol GCs exhibit a higher background spike rate and thus can respond more linearly to contrast changes than their Off-type counterparts. Here, we show that a calcium-permeable AMPA receptor (CP-AMPAR) antagonist blocks background spiking and sustained light-evoked firing in On-type GCs while preserving transient light responses. These effects are selective for On-GCs and are occluded by a gap-junction blocker suggesting involvement of AII amacrine cells (AII-ACs). Direct recordings from AII-ACs, cobalt uptake experiments, and analyses of transcriptomic data confirm that CP-AMPARs are expressed by primate AII-ACs. Overall, our data demonstrate that under some background light levels, CP-AMPARs at the rod bipolar to AII-AC synapse drive sustained signaling in On-type GCs and thus contribute to the more linear contrast signaling of the primate On- versus Off-pathway.

• The overall structure of calmodulin is not affected by the G113R (G114R) mutation, but the local geometry around the mutation site changes. • The G113R (G114R) mutation stabilizes calmodulin in the apo-form but destabilizes the Ca-bound form. • Stabilization of apo/calmodulin leads to a significant loss of structural flexibility. • Changes in the intramolecular dynamics of calmodulin may lead to cardiac arrhythmia. Mutations in the small, calcium-sensing, protein calmodulin cause cardiac arrhythmia and can ultimately prove lethal. Here, we report the impact of the G113R variant on the structure and dynamics of the calmodulin molecule, both in the presence and in the absence of calcium. We show that the mutation introduces minor changes into the structure of calmodulin and that it changes the thermostability and thus the degree of foldedness at human body temperature. The mutation also severely impacts the intramolecular mobility of calmodulin, especially in the apo form. Glycine 113 acts as an alpha-helical C-capping residue in both apo/ - and Ca2+/calmodulin, but its exchange to arginine has very different effects on the apo and Ca2+ forms. The majority of arrhythmogenic calmodulin variants identified affects residues in the Ca2+ coordinating loops of the two C-domain EF-Hands, causing a 'direct impact on Ca2+ binding'. However, G113R lies outside a Ca2+ coordinating loop and acts differently and more similar to the previously characterized arrhythmogenic N53I. Therefore, we suggest that altered apo/CaM dynamics may be a novel general disease mechanism, defining low-calcium target affinity – or Ca2+ binding kinetics – critical for timely coordination of essential ion-channels in the excitation-contraction cycle. [Display omitted] [ABSTRACT FROM AUTHOR]

Mutations in the small, calcium-sensing, protein calmodulin cause cardiac arrhythmia and can ultimately prove lethal. Here, we report the impact of the G113R variant on the structure and dynamics of the calmodulin molecule, both in the presence and in the absence of calcium. We show that the mutation introduces minor changes into the structure of calmodulin and that it changes the thermostability and thus the degree of foldedness at human body temperature. The mutation also severely impacts the intramolecular mobility of calmodulin, especially in the apo form. Glycine 113 acts as an alpha-helical C-capping residue in both apo/ - and Ca 2+ /calmodulin, but its exchange to arginine has very different effects on the apo and Ca 2+ forms. The majority of arrhythmogenic calmodulin variants identified affects residues in the Ca 2+ coordinating loops of the two C-domain EF-Hands, causing a 'direct impact on Ca 2+ binding'. However, G113R lies outside a Ca 2+ coordinating loop and acts differently and more similar to the previously characterized arrhythmogenic N53I. Therefore, we suggest that altered apo/CaM dynamics may be a novel general disease mechanism, defining low-calcium target affinity - or Ca 2+ binding kinetics - critical for timely coordination of essential ion-channels in the excitation-contraction cycle., Competing Interests: Declaration of Competing Interest MTO acted as expert witness for the defense in the 2022/2023 Inquiry into the convictions of Kathlen Megan Folbigg (https://2022folbigginquiry.dcj.nsw.gov.au/). CVH, NMP and HI are employees of private companies., (Copyright © 2023 The Authors. Published by Elsevier Ltd.. All rights reserved.)