PMC

MCU-i4 and MCU-i11 Negatively Modulate MCU Activity in a Small-Scale Validation Assay
(A) Agonist-induced mitochondrial calcium uptake in intact HeLa cells. Cells were stimulated with 100 μM histamine and treated with 10 μM of each compound in 0.1% DMSO. Left: mean [Ca²⁺]mt peaks are shown. Right: representative traces of mitochondrial calcium uptake are shown.
(B) Agonist-induced cytosolic calcium transients in intact HeLa cells. Cells were stimulated with 100 μM histamine and treated with 10 μM of each compound. Left: mean cytosolic calcium peaks are shown. Right: representative traces of cytosolic calcium transients are shown.
(C) Mitochondrial calcium uptake in permeabilized HeLa cells. A buffer mimicking the cytosolic ionic composition (IB) supplemented with either 100 μM EGTA (IB/EGTA) or 3 μM [Ca²⁺] was used. HeLa cells were permeabilized by a 1-min perfusion with 100 μM digitonin (in IB/EGTA) during luminescence measurements. Left: mean mitochondrial [Ca²⁺] speed is shown. Right: representative traces of mitochondrial calcium uptake are shown.
(D) Mitochondrial calcium uptake in intact HeLa cells upon long-term compound incubation.
(E) Agonist-induced mitochondrial calcium uptake in intact MEFs. Cells were stimulated with 100 μM ATP and treated with 10 μM compound.
(F) Agonist-induced mitochondrial calcium uptake in MDA-MB-231 cells upon treatment with 10 μM of each compound. Cells were stimulated with 100 μM ATP.
(G) Agonist-induced mitochondrial calcium uptake in HEK293T cells upon treatment with 10 μM of each compound. Cells were stimulated with 100 μM ATP.
(H) Δψ measurements in HeLa cells upon treatment with 10 μM of each compound at different time points.
Data are presented as mean ± SD. ^∗p < 0.05; ^∗∗p < 0.01; ^∗∗∗p < 0.001; one-way ANOVA except Kruskal-Wallis for (C).
See also .

A, Replacing external sodium with lithium (light gray column) caused a reversible increase in cytosolic calcium levels while a 10s application of 50mM KCl (arrow) depolarized the taste cells and opened VGCCs. The calcium influx through the VGCCs generated a large increase in cytosolic calcium that slowly returned to baseline levels. B, The integrated area under the 50mM KCl + lithium elevation was significantly larger than the combination of the integrated areas for the calcium elevations that occurred when 50mM KCl and lithium were applied individually (***p<0.001). Adding the area of the 50mM KCl-evoked calcium response (light gray column) to the integrated area of the calcium response when NCXs were inhibited (dark gray column) was only 79% of the integrated area of the calcium response that was generated when VGCCs were open while NCXs were inhibited (striped column). C, Replacing external sodium with lithium (Li) caused a reversible increase in cytosolic calcium levels while a 30s application of the bitter compound, 10mM denatonium benzoate (D) caused calcium release from internal stores. When bitter receptors were activated while NCXs were inhibited, the cytosolic calcium response increased. D, The integrated area under the lithium + denatonium response (Li + Den, striped column) was approximately equal to the combination of the integrated areas for the calcium signals that occurred when denatonium (light gray column) and lithium (dark gray column) were applied individually. Reprinted from Szenbenyi et al., (2010).

Effect of Compound 1 on CCK-stimulated intracellular calcium responses at human CCK1R and CCK2R. Shown in (A) is the chemical structure of Compound 1 and the impact of 10 µM of this compound on intracellular calcium responses to CCK-8 in CHO-CCK1R cells that express high levels of this receptor. Compound 1 shifted the CCK-8 concentration–response curve to the left, acting as a PAM (). (B) shows that Compound 1 did not directly stimulate an intracellular calcium response in the CHO-CCK1R cells when used in concentrations as high as 10 µM, and stimulated submaximal responses when used in 32 µM and 100 µM concentrations, while even these very high concentrations of Compound 1 did not elicit significant intracellular calcium responses at the non-receptor-bearing parental CHO cells. (C) shows that under similar conditions, Compound 1 had no modulatory effect on the ability of CCK-8 to stimulate intracellular calcium responses in CHO-CCK2R cells expressing the highly related type 2 CCK receptor (). (D, E) illustrate the ability of various concentrations of Compound 1 to affect concentration–response curves for CCK-8 and CCK-OPE to stimulate intracellular calcium responses in CHO-CCK1R cells. These assays were also performed with very high concentrations of Compound 1 to allow calculation of allosteric constants. Under these conditions, PAM effects of this compound are obvious, with most marked shifts observed for the partial agonist, CCK-OPE. Values are expressed as percentages of the intracellular calcium responses to maximal stimulation achieved by 0.1 mM ATP and data points represent the means ± S.E.M. of data from a minimum of three independent experiments performed in duplicate. (F, G) show curves reflecting fluorescent CCK binding association and dissociation in the absence (F) and presence (G) of 10 μM Compound 1. The off-rate of bound peptide is significantly slowed in the presence of Compound 1. Values reflect means ± S.E.M. of data from a minimum of three independent experiments.

Signaling pathways involved in the regulation of P. falciparum erythrocyte invasion: (A) The release of intracellular calcium is required for efficient invasion to proceed. BAPTA (an extracellular calcium chelator) has no effect on invasion, while BAPTA A/M (a membrane-permeable calcium chelator) shows a dose-dependent inhibition of invasion. (B) The release of intracellular calcium required for invasion is not via the Phospholipase-C/IP3 dependent pathway. U73122 (a PLC-specific inhibitor) has no specific effect on invasion, as the decrease in invasion efficiency using this compound is less significant than that seen using a non-specific analog of this compound, U73343. 2-APB, an inhibitor of IP3-dendent calcium release, also has no effect on invasion. (C) The cADP-Ribose dependent pathway for calcium release is involved in the regulation of invasion. 8Br-cADP-Ribose and Dantrolene are inhibitors of cADP-Ribose dependent calcium release, and each of these compounds causes a significant decrease in RBC invasion. (D) The release of intracellular calcium that is required for invasion must itself be well regulated. The calcium ionophore, A23187, artificially induces intracellular calcium release and causes a significant reduction in invasion efficiency at low concentrations.