ca ion selective electrode ise 9720bnwp  (Thermo Fisher)

Journal: bioRxiv

Article Title: State-specific inhibition of NMDA receptors by memantine depends on intracellular calcium and provides insights into NMDAR channel blocker tolerability

doi: 10.1101/2024.04.01.587624

Figure Lengend Snippet: A , Overlay of WT GluN1/2A receptor currents used to measure memantine (Mem) concentration-inhibition curves during application of glutamate (Glu; black bar) and memantine (red bars) For visualization of differences in inhibition, only traces in conditions of [Ca 2+ ] i < 1 nM (gray), [Ca 2+ ] i = 1 μM (teal), and [Ca 2+ ] i = 10 μM (blue) are shown. [Ca 2+ ] e = 0.1 mM to prevent Ca 2+ influx from altering [Ca 2+ ] i . Currents are normalized to steady state current measured in 0 memantine. B , Memantine concentration-inhibition curves for [Ca 2+ ] i = 10 μM (blue), 1 μM (teal), 100 nM (light blue), 10 nM (gold), and < 1 nM (gray). Lines depict fit of Hill equation to data. C , Summary of memantine IC 50 values measured at the indicated [Ca 2+ ] i (< 1 nM Ca 2+ : IC 50 = 2.76 ± 0.27 μM, n = 9; 10 nM Ca 2+ : IC 50 = 1.93 ± 0.07 μM, n = 6; 100 nM Ca 2+ : IC 50 = 1.76 ± 0.12 μM, n = 5; 1 μM Ca 2+ : IC 50 = 1.07 ± 0.04 μM, n = 4; 5 μM Ca 2+ : IC 50 = 0.69 ± 0.06 μM, n = 5; 10 μM Ca 2+ : IC 50 = 0.69 ± 0.05 μM, n = 5; 50 μM Ca 2+ : IC 50 = 0.70 ± 0.06 μM, n = 5). ANOVA with Sidak’s post hoc test. *p < 0.05, **p < 0.01, ****p < 0.0001. D, Curve describing the effect of [Ca 2+ ] i on memantine IC 50 . Line depicts fit of to data. Memantine becomes more potent (IC 50 decreases) as [Ca 2+ ] i increases. Dashed line at memantine IC 50 = 0.69 μM depicts minimum IC 50 . Dotted lines and open circle show the [Ca 2+ ] i required to induce a half-maximal effect on memantine IC 50 ([Ca 2+ ] i = 54 nM). E , Mg 2+ concentration-inhibition curves for [Ca 2+ ] i < 1 nM (gray) and [Ca 2+ ] i = 10 μM (blue). Lines depict fit of to data. F , Summary of Mg 2+ IC 50 values measured at [Ca 2+ ] i of < 1 nM (gray; IC 50 = 39.9 ± 3.3 nM, n = 6) and 10 μM (blue; IC 50 = 38.3 ± 2.7 nM, n = 7). 2-tailed Student t-test, p = 0.72. G , Ketamine concentration-inhibition curves for [Ca 2+ ] i < 1 nM (gray) and [Ca 2+ ] i = 10 μM (blue). Lines depict fit of to data. H , Summary of ketamine IC 50 values measured at [Ca 2+ ] i of < 1 nM (gray; IC 50 = 0.82 ± 0.04, n = 6) and 10 μM (blue; IC 50 = 0.78 ± 0.03 μM, n = 7). 2-tailed Student t-test, p = 0.46. I , Overlay of WT GluN1/2A receptor currents used to measure effect of [Ca 2+ ] i on desensitization; recordings shown are same as A, with currents instead normalized to peak response to Glu. J , Curve describing the effect of [Ca 2+ ] i on desensitization. Line depicts fit of to data, dashed line at I SS /I Peak = 0.505 depicts maximum desensitization, dotted lines and open circle show [Ca 2+ ] i required to induce a half-maximal effect on desensitization ([Ca 2+ ] i = 82 nM). For C , F , and H , points represent values from individual cells, bars and error bars depict mean ± SEM. For B , D , E , G , and J , data are depicted as mean ± SEM and some error bars are smaller than symbols. [Ca 2+ ] T and buffer used for each internal solution are given in , and [B] T for each buffer is given in .

Article Snippet: All measurements of [Ca 2+ ] F were made at 25° C using a Ca 2+ -selective combination electrode (Orion 9720BNWP, ThermoFisher) and a pH meter in mV mode (Accument AR15, ThermoFisher).

Techniques: Concentration Assay, Inhibition

Journal: bioRxiv

Article Title: State-specific inhibition of NMDA receptors by memantine depends on intracellular calcium and provides insights into NMDAR channel blocker tolerability

doi: 10.1101/2024.04.01.587624

Figure Lengend Snippet: A , Overlay of current traces used to measure memantine concentration-inhibition curves from native NMDARs in cultured cortical neurons at [Ca 2+ ] i < 1 nM (gray) and [Ca 2+ ] i = 50 μM (red). Traces are normalized to steady-state current before application of memantine to facilitate comparison of inhibition between conditions. Black bar depicts Glu application; red bars depict memantine applications; blue bar depicts application of the selective GluN1/2B antagonist CP101,606 (1 μM). B , Memantine concentration-inhibition curves measured with [Ca 2+ ] i < 1 nM (gray) and [Ca 2+ ] i = 50 μM (red) in DIV 15-22 cultured cortical neurons. Curves and fractional current values measured in the absence of CP101,606 are depicted with dashed lines and open circles; curves and fractional current values measured in the presence of CP101,606 are depicted with solid lines and circles. C , Summary of memantine IC 50 values measured at [Ca 2+ ] i of < 1 nM and 50 μM in the presence and absence of CP101,606. Memantine potency was significantly lower at [Ca 2+ ] i < 1 nM than [Ca 2+ ] i = 50 μM in the absence (2.63 ± 0.12 μM vs 1.48 ± 0.12 μM) and presence (3.46 ± 0.34 μM vs 1.56 ± 0.0.08 μM) of CP101,606. CP101,606 weakened memantine potency with [Ca 2+ ] i < 1 nM (3.46 ± 0.34 μM vs 2.63 ± 0.12 μM). One-way ANOVA with Tukey’s post hoc test.. D , Summary of ketamine IC 50 values measured at [Ca 2+ ] i of < 1 nM (gray; IC 50 = 1.44 ± 0.14), and 50 μM (red; IC 50 = 1.55 ± 0.09 μM in the presence of CP101,606). E , F , Representative recordings from pyramidal cells in cortical slices depicting differences in memantine inhibition with intracellular solutions containing 10 mM BAPTA ( E ) or 50 μM free calcium ( F ). Responses to single pulses and pulse trains are shown on the left and right, respectiviely. Ticks above current traces depict pulse times. G , Comparision of memantine inhibition of single evoked responses in low and high intracellular calcium condtions. Inhibition was significantly stronger (I Mem /I control was lower) in conditions of high intracellular calcium (2-tailed Student t-test). H , Summary of memantine inhibition of responses evoked by pulse train in low and high intracellular calcium condtions. Inhibition was significantly stronger in conditions of high intracellular calcium and was stable across pulse number (2-way ANOVA with Tukey’s post hoc test). For B and H , data are depicted as mean ± SEM and some error bars are smaller than symbols. For C, D , G , points represent values from individual cells, bars and error bars depict mean ± SEM. *p < 0.05, **p < 0.01, ****p < 0.0001.

Article Snippet: All measurements of [Ca 2+ ] F were made at 25° C using a Ca 2+ -selective combination electrode (Orion 9720BNWP, ThermoFisher) and a pH meter in mV mode (Accument AR15, ThermoFisher).

Techniques: Concentration Assay, Inhibition, Cell Culture, Comparison, Control

Journal: bioRxiv

Article Title: State-specific inhibition of NMDA receptors by memantine depends on intracellular calcium and provides insights into NMDAR channel blocker tolerability

doi: 10.1101/2024.04.01.587624

Figure Lengend Snippet: A , B Overlay of GluN1/2A ( A ) and GluN1/2B ( B ) receptor responses with [Ca 2+ ] i = 50 μM recorded at 5 (light red) and 15 (red) min after break-in. Currents are normalized to I Peak . C , D , GluN1/2A ( C ) and GluN1/2B ( D ) receptor desensitization as a function of duration of exposure to [Ca 2+ ] i = 50 μM. Desensitization greatly increases with duration of exposure to high [Ca 2+ ] i (Repeated measures one-way ANOVA with test for linear trend (***p < 0.001, ****p < 0.0001). E , F , Memantine IC 50 for GluN1/2A ( E ) and GluN1/2B ( F ) receptors plotted as a function of duration of exposure to [Ca 2+ ] i = 50 μM. Memantine potency is not related to duration of exposure to high [Ca 2+ ] i (Repeated measures one-way ANOVA with test for linear trend (p = 0.29). G , Overlay of GluN1/2B receptor responses recorded at 5 (light red) and 15 (red) min after break-in with [Ca 2+ ] i = 50 μM and kinase activity inhibited. Currents are normalized to I Peak . H , GluN1/2B receptor desensitization as a function of duration of exposure to [Ca 2+ ] i = 50 μM with kinase activity inhibited. Kinase inhibition removed the dependence of desensitization on duration of exposure to high [Ca 2+ ] i (5 min: I ss /I Peak = 0.67 ± 0.01; 10 min: I ss /I Peak = 0.67 ± 0.03; 15 min: I ss /I Peak = 0.63 ± 0.04). Repeated measures one-way ANOVA with test for linear trend (p = 0.07). Points in C - F , H represent values from individual cells, bars and error bars depict mean ± SEM. [Ca 2+ ] T and buffer used for each internal solution are given in , and [B] T for each buffer is given in .

Article Snippet: All measurements of [Ca 2+ ] F were made at 25° C using a Ca 2+ -selective combination electrode (Orion 9720BNWP, ThermoFisher) and a pH meter in mV mode (Accument AR15, ThermoFisher).

Techniques: Activity Assay, Inhibition

Journal: bioRxiv

Article Title: State-specific inhibition of NMDA receptors by memantine depends on intracellular calcium and provides insights into NMDAR channel blocker tolerability

doi: 10.1101/2024.04.01.587624

Figure Lengend Snippet: Measured [Ca 2+ ]F values in Ca 2+ Buffer solutions.

Article Snippet: All measurements of [Ca 2+ ] F were made at 25° C using a Ca 2+ -selective combination electrode (Orion 9720BNWP, ThermoFisher) and a pH meter in mV mode (Accument AR15, ThermoFisher).

Techniques:

Journal: bioRxiv

Article Title: State-specific inhibition of NMDA receptors by memantine depends on intracellular calcium and provides insights into NMDAR channel blocker tolerability

doi: 10.1101/2024.04.01.587624

Figure Lengend Snippet: A-D , Representative current traces of WT GluN1/2A ( A , B ) and GluN1ΔCTD/2A receptor responses to 1 mM Glu (black bars) after inter-application intervals of decreasing duration in the absence ( A , C ) and presence ( B , D ) of 3 μM memantine (red bars). Dotted line is placed at I Peak after the 200 s inter-application interval to allow visualization of differences between peaks. E , F , Exponential fits to time course of recovery from desensitization (RfD). Symbols depict normalized I Peak values for WT receptors ( E ) or GluN1ΔCTD/2A receptors ( F ). G , Summary and comparison of time constant of RfD values (WT Control τ = 4.66 ± 0.95 s, n = 8; WT Mem τ w = 44.56 ± 3.46 s; GluN1ΔCTD Control τ = 2.58 ± 0.32 s; n = 4; GluN1ΔCTD Mem τ = 2.58 ± 0.32 s; n = 4). ANOVA with Tukey’s post hoc test. Intracellular solutions contained 10 mM BAPTA and no added CaCl 2 . H , Representative GluN1ΔCTD/2A receptor currents used to measure memantine concentration-inhibition relations in conditions of [Ca 2+ ] i = <1 nM (gray) and 5 μM (blue) in 0.1 mM Ca 2+ e , normalized to I SS . Inset depicts overlay of GluN1ΔCTD/2A receptor currents normalized to I Peak . I , Summary of memantine IC 50 values for WT GluN1/2A and GluN1ΔCTD/2A receptors in conditions shown in H . GluN1 CTD truncation ablates the effect of [Ca 2+ ] I on memantine IC 50 (for GluN1ΔCTD/2A receptors, [Ca 2+ ] i < 1 nM: 2.13 ± 0.27 μM, n = 4; [Ca 2+ ] i = 5 μM: 2.18 ± 0.34 μM, n = 4). 2-way ANOVA (interaction p < 0.001) with Tukey post hoc test. J , Comparison of I SS /I Peak values of GluN1ΔCTD/2A receptors with [Ca 2+ ] i <1 nM and [Ca 2+ ] i = 5 μM. No difference in desensitization was observed (Student t-test). In E - F , data are depicted as mean ± SEM and some error bars are smaller than symbols; in G , I , and J , points represent values from individual cells, bars and error bars depict mean ± SEM ***p < 0.001, ****p < 0.0001. [Ca 2+ ] T and buffer used for each internal solution are given in , and [B] T for each buffer is given in .

Article Snippet: All measurements of [Ca 2+ ] F were made at 25° C using a Ca 2+ -selective combination electrode (Orion 9720BNWP, ThermoFisher) and a pH meter in mV mode (Accument AR15, ThermoFisher).

Techniques: Comparison, Control, Concentration Assay, Inhibition

Journal: bioRxiv

Article Title: State-specific inhibition of NMDA receptors by memantine depends on intracellular calcium and provides insights into NMDAR channel blocker tolerability

doi: 10.1101/2024.04.01.587624

Figure Lengend Snippet: A-D , Overlay of current traces used to measure memantine concentration-inhibition curves for indicated NMDAR subtype in cells with [Ca 2+ ] i < 1 nM (gray) and [Ca 2+ ] i = 10 μM (blue). [Ca 2+ ] e = 0.1 mM. Traces are normalized to I SS before application of memantine to facilitate comparison of inhibition between [Ca 2+ ] i conditions. Black bar depicts Glu application; red bars depict memantine applications. Insets depict overlay of responses to 1 mM Glu normalized to I Peak in the absence of memantine, which were used to measure I SS /I Peak . E-H , Concentration-inhibition curves for indicated NMDAR subtype and [Ca 2+ ] i . Points and error bars show mean ± SEM. Some error bars are smaller than points. I-L , Summary of IC 50 values for indicated receptor subtype and [Ca 2+ ] i . Inhibition of GluN1/2A receptors by memantine depends on [Ca 2+ ] i . GluN1/2B, GluN1/2C, and GluN1/2D receptor inhibition is unaffected by [Ca 2+ ] i . M-P , Summary of I SS /I Peak values for indicated receptor subtype and [Ca 2+ ] i . GluN1/2A and GluN1/N2B receptors show CDD. Desensitization of GluN1/2C and GluN1/2D receptors does not depend on [Ca 2+ ] i . Points represent values from individual cells, bars and error bars show mean ± SEM; 2-tailed Student t-test, **p < 0.01, ***p < 0.001,****p < 0.0001. [Ca 2+ ] T and buffer used for each internal solution are given in , and [B] T for each buffer is given in . Memantine IC 50 and I SS /I Peak numeric values for all subtypes are summarized in .

Article Snippet: All measurements of [Ca 2+ ] F were made at 25° C using a Ca 2+ -selective combination electrode (Orion 9720BNWP, ThermoFisher) and a pH meter in mV mode (Accument AR15, ThermoFisher).

Techniques: Concentration Assay, Inhibition, Comparison