Article Figures & Data
Figures
- Figure 1.
Modulator-activated IMI measurement and quantification. A, Proctolin (1 μm)-induced IMI in LP neuron. Top, Voltage ramp protocol. Middle, Current in control ramp (black trace) and at the peak of the response to proctolin (red trace). Bottom, Difference current (blue trace) obtained by subtracting control currents from currents measured in proctolin. Only currents evoked during descending voltage ramps were considered (blue trace). B, I–V curves of IMI in normal saline solution (13 mm CaCl2, black trace) and low-calcium saline solution (2 mm CaCl2, blue trace). Changes in slope between −75 and −20 mV are used as a measure of IMI voltage dependence. Amplitude at −15 mV is taken as a measure of IMI activation. Note that IMI in normal saline solution is close to maximal at this voltage, and there is minimal difference between normal calcium and low-calcium conditions.
- Figure 2.
The effect of calmodulin inhibitors on IMI voltage dependence. Left, Proctolin-induced IMI at different concentrations of W7. Ai, Representative I–V curves of a W7 experiment. Aii, Averaged I–V curves across W7 experiments. Aiii, Quantification of W7 data. A one-way repeated-measures ANOVA showed that W7 changed the proctolin-induced IMI slope (F(4,19) = 15.972, p = 6.96 × 10−6j). Error bars indicate the SEM. Tukey’s test; *p < 0.05; ***p < 0.001. Right, Proctolin-induced IMI in different concentrations of calmidazolium (CDZ). Bi, Representative I–V curves. Bii, Average I–V curves from all calmidazolium experiments. Biii, Quantification of all calmidazolium data. A one-way repeated-measures ANOVA showed that calmidazolium significantly altered IMI slope (F(3,9) = 4.846, p = 0.028n). However, no significant post hoc pairwise differences were observed. Tukey’s post hoc test, p < 0.05. Error bars indicate the SEM. Data are from LP neurons.
- Figure 3.
The ryanodine antagonist dantrolene reduces IMI voltage dependence. A, Representative I–V curve of the proctolin-induced IMI before (black trace) and after (red trace) application of 3.33 μm dantrolene. B, Averaged I–V curves of all dantrolene experiments. C, Quantification of dantrolene data shown in B. A paired two-tailed Student’s t test showed that dantrolene significantly increased proctolin-induced IMI slope (t(5) = −4.230, p = 0.008t). *p < 0.05. Error bars indicate the SEM. Recordings are from LP neurons.
- Figure 4.
The CaMKII inhibitor KN-93 reduces IMI voltage dependence. Proctolin-induced IMI at different concentrations of KN-93. For statistical analysis, KN-93 was grouped into a low-dose (2-5 μm) and a high-dose (10-20 μm) group, in addition to a control group (0 μm). A, Representative I–V curves for a KN-93 experiment. B, Averaged I–V curves for all KN-93 experiments. C, Quantification of data shown in B. A one-way repeated-measures ANOVA showed that KN-93 significantly increased proctolin-induced IMI slope (F(2,5) = 46.239, p = 5.96 × 10−4w). Recordings are from LP neurons.
- Figure 5.
The Gβγ-subunit inhibitor gallein increases IMI slope. A, Representative I–V curves of proctolin-induced IMI in gallein. B, Averaged I–V curves of proctolin-induced IMI for all gallein experiments. C, Quantification of the data shown in A. B, A one-way repeated-measures ANOVA showed that gallein significantly increased proctolin-induced IMI slope (F(2,16) = 4.445, p = 0.029y). Error bars indicate the SEM. Tukey’s test, *p < 0.05. Recordings are from LP neurons.
- Figure 6.
The specific CaSR antagonist NPS-2143 increases IMI slope in a normal calcium condition in LP neurons. A, Representative I–V curves showing the effect of NPS-2143 (NPS) at different concentrations on proctolin-induced IMI. B, Averaged I–V curves of all NPS-2143 experiments. C, Quantification of all NPS-2143 data. A one-way repeated-measures ANOVA showed that NPS-2143 significantly altered proctolin-induced IMI slope (F(4,22) = 3.314, p = 0.029aa). Error bars indicate the SEM. Tukey’s test, *p < 0.05.
- Figure 7.
The MLCK inhibitor ML-7 reduces the voltage dependence of IMI in LP neurons. A, Representative I–V traces of proctolin-induced IMI in the presence of various concentrations of ML-7. B, Averaged I–V traces for all ML-7 experiments. C, A one-way repeated-measures ANOVA showed that ML-7 increased proctolin-induced IMI slope (F(3,26) = 7.503, p = 8.92 × 10−4ac). Error bars indicate the SEM. Tukey’s test: *p < 0.05; **p < 0.01; ***p < 0.001.
- Figure 8.
The endocytosis inhibitor dynasore prevents W7-induced increases in IMI slope in LP neurons. CCAP-induced IMI was measured before (black) and after exposure to the following: 33 µm W7 for 45-65 min (red); dynasore for 45-80 min (blue); 20 min in 33 µm dynasore, then 45-60 min in 33 µm dynasore plus 33 µm W7 (green). A, Representative I–V curves of W7 effect (top) and dynasore effect (bottom). B, Average I–V curves of all experiments. C, Average slope of CCAP-induced IMI from data in B and two-way ANOVA for the effects of factors dynasore and W7 on slope (W7: F(1,32) = 14.934, p = 5.12 × 10−4; dynasore: F(1,32) = 4.317, p = 0.046; Interaction: F(1,32) = 2.109, p = 0.156ae). Post hoc Tukey’s comparisons on slope: (1) W7 only vs control (p < 0.001); (2) dynasore only vs control (p = 0.624); (3) dynasore only vs dynasore plus W7 (p = 0.129); and (4) W7 only vs W7 plus dynasore (p = 0.03). Post hoc Tukey’s test: *p < 0.05; ***p < 0.001.
- Figure 9.
The CaSR agonist R568 increases proctolin-induced IMI slope in LP neurons. Proctolin-induced IMI was measured in the presence (red) or absence (black) of 10 µm CaSR agonist R568 in either 13 mm CaCl2 (solid) or 2 mm CaCl2 (striped). A, Left, Representative I–V curves of proctolin-induced IMI in 13 mm CaCl2 (black solid), 2 mm CaCl2 (black dotted), and 13 mm CaCl2 (green solid) after a 1 h wash. Right, Representative I–V curves of proctolin-induced IMI in the presence of 10 µm CaSR agonist R568 in normal (13 mm) calcium (red solid), low (2 mm) calcium (red dotted), and then in normal calcium after 1 h wash from R568 (green solid). B, Left, Averaged I–V traces of all proctolin-induced IMI experiments in normal calcium in the presence (red solid) or absence (black solid) of 10 µm R568. Right, Averaged I–V traces of all proctolin-induced IMI experiments in a low-calcium condition in the presence (red dotted) or absence (black dotted) of 10 µm R568. C, Quantification of IMI slope. Two-way ANOVA for factors R568 and calcium showing significant changes in proctolin-induced IMI slope (calcium: F(1,20) = 8.560, p = 0.008; R568: F(1,20) = 9.295, p = 0.006; interaction: F(1,20) = 0.0324, p = 0.859ag). Error bars indicate the SEM. Tukey’s test: *p < 0.05; ***p < 0.001.
- Figure 10.
A model for CaSR-mediated regulation of IMI voltage dependence. According to this model, IMI channels are activated by a neuropeptide receptor using a pathway (not shown here) independent from the depicted voltage dependence pathway. The voltage dependence of IMI is regulated by G-protein-coupled CaSRs. Calmodulin (CaM) stabilizes the receptor on the membrane, and inhibitors of calmodulin lead to CaSR endocytosis. Green boxes and blunt-ended lines show agents that inhibit the indicated paths. Arrows indicate activating pathways. Intracellular calcium release via ryanodine receptors is part of the source for calmodulin activation, and both CaMKII and MLCK inhibit voltage dependence.
Tables
- Table 1:
Effect of proctolin application sequence on maximum amplitude and slope in normal calcium saline solution
Application 1 Application 2 Application 3 Application 4 Application 5 Ampl (nA) Slope (nS) Ampl (nA) Slope (nS) Ampl (nA) Slope (nS) Ampl (nA) Slope (nS) Ampl (nA) Slope (nS) (n = 7) (n = 7) (n = 7) (n = 7) (n = 7) (n = 7) (n = 7) (n = 7) (n = 5) (n = 5) −2.8 ± 0.6* −41 ± 10 −1.3 ± 0.3 −17 ± 4 −1.5 ± 0.3 −17 ± 5 −1.1 ± 0.3 −12 ± 3 −1.0 ± 0.3 −12 ± 6 All data were obtained from LP neurons. Ampl, Amplitude.
*Tukey’s test, p < 0.05 vs application 2.
- Table 2:
Effect of proctolin application sequence on maximum amplitude and slope in low-calcium saline solution
Application 2 Application 3 Application 4 Application 5 Application 6 Ampl (nA) Slope (nS) Ampl (nA) Slope (nS) Ampl (nA) Slope (nS) Ampl (nA) Slope (nS) Ampl (nA) Slope (nS) (n = 3) (n = 3) (n = 3) (n = 3) (n = 3) (n = 3) (n = 3) (n = 3) (n = 3) (n = 3) −2.1 ± 0.9 +26 ± 10 −1.6 ± 0.7 +23 ± 11 −1.1 ± 0.6* +19 ± 5 −0.8 ± 0.6** +22 ± 6 −0.7 ± 0.5** +18±6 Data are reported as the mean ± SEM. First application was not included as it was performed in normal calcium conditions. All data obtained from LP neurons. Ampl, Amplitude.
*Tukey’s test, p < 0.05 vs application 2 of same calcium condition. **Tukey test’s, p < 0.01 vs application 2 of same calcium condition.
Drug name [Drug] [Ca] Neuromodulator n IMI slope (nS) W7 0 µm 13 mm Proctolin 12 −21.7 ± 3.7 0.1 µm 13 mm Proctolin 5 −13.5 ± 4.2 1 µm 13 mm Proctolin 6 −11.2 ± 2.4* 10 µm 13 mm Proctolin 6 −11.4 ± 3.9** 100 µm 13 mm Proctolin 6 +13.1 ± 3.4*** Calmidazolium 0 µm 13 mm Proctolin 6 −20.4 ± 10.6 0.1 µm 13 mm Proctolin 2 −13.7 ± 11.1 0.3 µm 13 mm Proctolin 6 +1.6 ± 14 0.0 µm 13 mm Proctolin 4 +4.3 ± 4.9 Dantrolene 0 µm 13 mm Proctolin 6 −8.1 ± 3.2 3.3 µm 13 mm Proctolin 6 +1.6 ± 1.9** CALP1 0 µm 2 mm Proctolin 11 +18.2 ± 4.9 1 µm 2 mm Proctolin 4 +10.2 ± 6.4 10 µm 2 mm Proctolin 4 +28.2 ± 3.8 50 µm 2 mm Proctolin 2 +23.5 ± 9.4 KN-93 0 µm 13 mm Proctolin 7 −21 ± 4.2 Low dose 13 mm Proctolin 5 +0.8 ± 5.8 High dose 13 mm Proctolin 2 +16.6 ± 2.2 Pertussis toxin 0 µg/ml 13 mm Proctolin 7 −3.1 ± 2.9 0 µg/ml 2 mm Proctolin 6 +14.4 ± 3.1 10 µg/ml 13 mm Proctolin 7 +1.5 ± 2.9 10 µg/ml 2 mm Proctolin 6 +21.5 ± 3.1 GTPγS 0 mm 13 mm Proctolin 9 −2.2 ± 2.9 0 mm 2 mm Proctolin 8 +11.5 ± 3.1 10 mm 13 mm Proctolin 5 −6.7 ± 4.0 10 mm 2 mm Proctolin 4 +15.2 ± 4.4 Gallein 0 µm 13 mm Proctolin 9 −5.4 ± 1.6 1 µm 13 mm Proctolin 9 +1.5 ± 1.9* 3 µm 13 mm Proctolin 9 +0.7 ± 2.8 ML-7 0 µm 13 mm Proctolin 12 −4.2 ± 1.5 0.1 µm 13 mm Proctolin 12 +1.7 ± 2.3* 1 µm 13 mm Proctolin 10 +4.5 ± 1.4*** 10 µm 13 mm Proctolin 7 +3.1 ± 2.4* Dynasore, W7 0, 0 µm 13 mm CCAP 18 −11.3 ± 1.3 0, 33 µm 13 mm CCAP 6 −0.4 ± 2.3*** 33, 0 µm 13 mm CCAP 6 −12.6 ± 2.3 33, 33 µm 13 mm CCAP 6 −7.7 ± 2.3† R568 0 µm 13 mm Proctolin 10 −0.7 ± 4.5 0 µm 2 mm Proctolin 6 +16.5 ± 5.9 10 µm 13 mm Proctolin 4 +17.3 ± 7.2* 10 µm 2 mm Proctolin 4 +36.9 ± 7.2‡ Data are reported as the mean ± SEM. All data were obtained from LP neurons.
Tukey’s test/t test p value vs control: *p < 0.05, **p < 0.01, ***p < 0.001; †p < 0.05 vs W7 alone; ‡p < 0.05 vs low-calcium control.
Letter, experiment name Data structure (distribution) Type of test Power (α = 0.05) a Low-calcium effect on RMP Normal Two-tailed paired t test 1.00 b Low-calcium effect on Rin Normal Two-tailed paired t test 0.84 c BSA effect on low calcium-induced depolarization Normal Two-tailed t test 1.00 d BSA effect on low calcium-induced effects of Rin Non-normal Mann–Whitney rank sum test 0.17* e Effect of low calcium on IMI amplitude Normal One-way ANOVA 0.30 f Effect of application number on IMI amplitude in normal calcium Normal One-way ANOVA, post hoc Tukey’s tests 0.81 g Effect of application number on slope in normal calcium Normal One-way ANOVA, post hoc Tukey’s tests 0.81 h Effect of application number on IMI amplitude in low calcium Normal Two-way repeated-measures ANOVA, post hoc Tukey’s tests Calcium = 0.05Application = 0.90Interaction = 0.59 i Effect of application number on slope in low calcium Normal Two-way repeated-measures ANOVA, post hoc Tukey’s tests Calcium = 0.94Application = 0.05Interaction = 0.13 j W7 effect on proctolin-induced IMI slope Normal One-way repeated-measures ANOVA, post hoc Tukey’s tests 1.00 k W7 effect on proctolin-induced IMI amplitude Normal One-way repeated-measures ANOVA, post hoc Tukey’s tests 0.54 l W7 effect on CCAP-induced IMI slope Normal Two-tailed paired t test 0.50 m W7 effect on CCAP-induced IMI amplitude Normal Two-tailed paired t test 0.05 n Effect of calmidazolium on proctolin-induced IMI slope Normal One-way repeated-measures ANOVA, post hoc Tukey’s tests 0.62 o Effect of calmidazolium effect on proctolin-induced IMI amplitude Normal One-way repeated-measures ANOVA, post hoc Tukey’s tests 0.78 p Effect of CALP1 (2 h) on proctolin-induced IMI slope Normal One-way ANOVA 0.07 q Effect of CALP1 (2 h) on proctolin-induced IMI amplitude Normal One-way ANOVA 0.05 r Effect of CALP1 (overnight) on proctolin-induced IMI slope Normal Two-tailed t test 0.10 s Effect of CALP1 (overnight) on proctolin-induced IMI amplitude Normal Two-tailed t test 0.10 t Dantrolene effect on proctolin-induced IMI slope Normal Two-tailed paired t test 0.91 u Dantrolene effect on proctolin-induced IMI amplitude Normal Two-tailed paired t test 0.77 w KN-93 effect on proctolin-induced IMI slope Normal One-way repeated-measures ANOVA, post hoc Tukey’s tests 1.00 x KN-93 effect on proctolin-induced IMI amplitude Normal One-way repeated-measures ANOVA, post hoc Tukey’s tests 1.00 y Effect of gallein on proctolin-induced IMI slope Normal One-way repeated-measures ANOVA, post hoc Tukey’s tests 0.56 z Effect of gallein on proctolin-induced IMI amplitude Normal One-way repeated-measures ANOVA, post hoc Tukey’s tests 0.13 aa Effect of NPS-2143 on proctolin-induced IMI slope Normal One-way repeated-measures ANOVA, post hoc Tukey’s tests 0.57 ab Effect of NPS-2143 on proctolin-induced IMI amplitude Normal One-way repeated-measures ANOVA, post hoc Tukey’s tests 0.06 ac ML7 effect on proctolin-induced IMI slope Normal One-way repeated-measures ANOVA, post hoc Tukey’s tests 0.95 ad ML7 effect on proctolin-induced IMI amplitude Normal One-way repeated-measures ANOVA, post hoc Tukey’s tests 0.71 ae Effect of dynasore on W7’s reduction of IMI voltage dependence Normal Two-way ANOVA, post hoc Tukey’s tests W7 = 0.99Dynasore = 0.55Interaction = 0.26 af Effect of dynasore on effect of W7 on IMI amplitude Normal Two-way ANOVA, post hoc Tukey’s tests W7 = 0.88Dynasore = 0.10Interaction = 0.17 ag R568 effect on proctolin-induced IMI slope Normal Two-way ANOVA, post hoc Tukey’s tests Calcium = 0.86R568 = 0.89Interaction = 0.10 ah R568 effect on proctolin-induced IMI amplitude Non-normal Two-way ANOVA, post hoc Tukey’s tests Calcium = 1.00R568 = 1.00Interaction = 0.70 All data obtained from LP neurons. RMP is resting membrane potential, Rin is input resistance.
*Post hoc power calculation performed for t test.
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