Activation mechanism of a neuromodulator-gated pacemaker ionic current

Abstract

The neuromodulator-gated current (I_MI) found in the crab stomatogastric ganglion is activated by neuromodulators that are essential to induce the rhythmic activity of the pyloric network in this system. One of these neuromodulators is also known to control the correlated expression of voltage-gated ionic currents in pyloric neurons, as well as synaptic plasticity and strength. Thus understanding the mechanism by which neuromodulator receptors activate I_MI should provide insights not only into how oscillations are initiated but also into how other processes, and currents not directly activated by them, are regulated. To determine what specific signaling molecules are implicated in this process, we used a battery of agonists and antagonists of common signal transduction pathways. We found that the G protein inhibitor GDPβS and the G protein activator GTPγS significantly affect I_MI amplitude, suggesting that its activation is mediated by G proteins. Interestingly, when using the more specific G protein blocker pertussis toxin, we observed the expected inhibition of I_MI amplitude but, unexpectedly, in a calcium-dependent fashion. We also found that antagonists of calcium- and calmodulin-associated signaling significantly reduce I_MI amplitude. In contrast, we found little evidence for the role of cyclic nucleotide signaling, phospholipase C (PLC), or kinases and phosphatases, except two calmodulin-dependent kinases. In sum, these results suggest that proctolin-induced I_MI is mediated by a G protein whose pertussis toxin sensitivity is altered by external calcium concentration and appears to depend on intracellular calcium, calmodulin, and calmodulin-activated kinases. In contrast, we found no support for I_MI being mediated by PLC signaling or cyclic nucleotides.NEW & NOTEWORTHY Neuronal rhythmic activity is generated by either network-based or cell-autonomous mechanisms. In the pyloric network of decapod crustaceans, the activation of a neuromodulator-gated pacemaker current is crucial for the generation of rhythmic activity. This current is activated by several neuromodulators, including peptides and acetylcholine, presumably via metabotropic receptors. We have previously demonstrated a novel extracellular calcium-sensitive voltage-dependence mechanism of this current. We presently report that the activation mechanism depends on intracellular and extracellular calcium-sensitive components.

Keywords: G proteins; calcium; calmodulin; pacemaker; signaling.

Fig. 1.

Proctolin-induced I_MI activation requires G proteins: effect of GDPβS. GDPβS (gray) or control solutions (black) were pressure injected in normal (13 mM CaCl₂; solid curves) or low extracellular (2 mM CaCl₂) calcium levels (dotted curves). A: averaged I-V curves of proctolin-induced I_MI in normal calcium. B: averaged I-V curves of proctolin-induced I_MI in 2 mM (low) extracellular calcium. C: quantification of the effect of GDPβS on I_MI: both GDPβs and calcium significantly altered I_MI amplitude at −15 mV [2-way ANOVA, calcium: F(1,30) = 5.99, P = 0.02; GDPβS: F(1,30) = 15.034, P = 5.3 × 10⁻⁴; interaction: F(1,30) = 0.376, P = 0.544]. Tukey comparisons: *P < 0.05; **P < 0.01; ns, not significant. Error bars are SE. Solid bars indicate normal extracellular Ca²⁺ (control); hatched bars indicate low calcium level.

Fig. 2.

Proctolin-induced I_MI activation requires G proteins: effect of GTPγS. GTPγS was pressure injected in a vehicle solution of 500 mM KCl + 20 mM TEA. Control pressure injections used the vehicle solution only. A: averaged I-V curves of proctolin-induced I_MI after pressure injection with either control (black) or 10 mM GTPγS (gray) solution in normal (13 mM) calcium. B: same as A but in saline containing low (2 mM) calcium. C: quantification of the effect of GTPγS on I_MI amplitude at −15 mV. A 2-way ANOVA showed that GTPγS, but not calcium, was capable of occluding I_MI amplitude [calcium: F(1,22) = 2.136, P = 0.158; GTPγS: F(1,22) = 13.562, P = 0.001; interaction: F(1,22) = 0.0817, P = 0.778]. Tukey post hoc test: *P < 0.05; ***P < 0.001; ns, not significant. Error bars are SE. Solid bars indicate normal extracellular Ca²⁺ (control); hatched bars indicate low calcium level.

Fig. 3.

The specific G protein inhibitor pertussis toxin (PTx-A) inhibits proctolin-induced I_MI. Control pressure injection solutions of 500 mM KCl + 20 mM TEA (black) or the same solution plus 10 μg/ml PTx-A (gray) were pressure injected in either normal (13 mM) or low (2 mM) extracellular calcium. All recording electrodes used 20 mM KCl + 0.6 M K₂SO₄. A: averaged I-V curves of proctolin-induced I_MI after pressure injection of control solution (black) or 10 μg/ml PTx-A solution (gray). B: same as A but in saline containing low (2 mM) calcium. C: quantification of effect of PTx-A on proctolin-induced I_MI amplitude. A 2-way ANOVA showed that both calcium and PTx-A were capable of altering I_MI amplitude at −15 mV [calcium: F(1,22) = 20.630, P = 1.61 × 10⁻⁴; PTx-A: F(1,22) = 11.735, P = 0.002; interaction: F(1,22) = 3.541, P = 0.073]. Post hoc Tukey test: *P < 0.05; ***P < 0.001; ns, not significant. Error bars are SE. Solid bars indicate normal extracellular Ca²⁺ (control); hatched bars indicate low calcium level.

Fig. 4.

Role of calmodulin in proctolin-induced I_MI amplitude. Proctolin-induced I_MI is shown at different concentrations of calmodulin inhibitors W7 (A) and calmidazolium (CDZ; B). A: quantification of W7 data. A 1-way repeated-measures ANOVA showed a small but significant reduction of proctolin-induced I_MI amplitude by W7 [F(4,19) = 3.24, P = 0.035]. B: proctolin-induced I_MI at different concentrations of CDZ. A 1-way repeated-measures ANOVA showed that CDZ reduced proctolin-induced I_MI amplitude [F(3,9) = 6.38, P = 0.013]. Error bars are SE. Tukey test: *P < 0.05.

Fig. 5.

The ryanodine receptor antagonist dantrolene inhibits proctolin-induced I_MI. Proctolin-induced I_MI is shown before (open bar) and after (filled bar) application of 3.33 μM dantrolene. A paired t-test showed that dantrolene significantly decreased proctolin-induced I_MI amplitude at −15 mV [t(5) = −3.502, P = 0.017]. Error bars are SE. *P < 0.05.

Fig. 6.

Kinase inhibitors reduce I_MI amplitude. A: proctolin-induced I_MI in different concentrations of KN-93. For statistical analysis, KN-93 was grouped into “low-dose” (2–5 μM range, average concentration 4.2 μM), and “high-dose” groups (10–20 μM range, average concentration 15 μM). A 1-way repeated-measures ANOVA showed that KN-93 significantly decreased proctolin-induced I_MI amplitude at −15 mV [KN-93: F(2,5) = 34.09, P = 0.001]. B: proctolin-induced I_MI at different concentrations of the MLCK inhibitor ML-7. A 1-way repeated-measures ANOVA showed that ML-7 decreased proctolin-induced I_MI amplitude at −15 mV [ML-7: F(3,26) = 4.468, P = 0.012]. Post hoc Tukey test: *P < 0.05; **P < 0.01; ns, not significant. Error bars are SE.

Fig. 7.

Role of the calcium sensing receptor (CaSR) in I_MI amplitude. Proctolin-induced I_MI in control (black) or in the presence of the CaSR agonist R568 (gray) was measured in either 13 mM (solid bars) or 2 mM CaCl₂ saline (hatched bars). A two-way ANOVA for factors R568 and calcium showed significant changes in proctolin-induced I_MI amplitude at −15 mV [calcium: F(1,20) = 18.301, P = 3.67 × 10⁻⁴; R568: F(1,20) = 23.447, P = 9.9 × 10⁻⁵; interaction: F(1,20) = 5.962, P = 0.024]. Post hoc Tukey test: ***P < 0.001; ns, not significant. Error bars are SE.

Fig. 8.

Proposed mechanism of activation for proctolin-induced I_MI. According to this model, I_MI channels are activated by a neuropeptide GPCR using a pertussis toxin-sensitive pathway that is independent from a different GPCR-mediated voltage-dependence pathway (not shown; cf. Gray and Golowasch 2016). Calmodulin (CaM) via calmodulin-dependent kinases CamKII and MLCK, activated by exogenous calcium entering the cell via either the I_MI channels themselves (shown) or voltage-dependent calcium channels (not shown), amplifies the activation. This is aided by a further increase in intracellular calcium level due to calcium-induced calcium release mediated by RyRs. Blunt-ended lines show agents that inhibit the indicated paths; arrows indicate activating agents and pathways. ER, endoplasmic reticulum; ProcR, proctolin receptor; RyR ryanodine receptor.