Differential Regulation of Two Distinct Voltage-Dependent Sodium Currents by Group III Metabotropic Glutamate Receptor Activation in Insect Pacemaker Neurons

Abstract

Using whole cell patch-clamp technique and immunocytochemistry on adult dorsal unpaired median (DUM) neurons isolated from the cockroach Periplaneta americana CNS, we reported the characterization of a native mGluR, sharing pharmacological properties with vertebrate metabotropic glutamate receptor III (mGluRIII) that regulated voltage-dependent sodium current (I(Na)). The global I(Na) was dissociated by means of l-glutamate sensitivity, deactivation time constant, voltage dependence of activation and inactivation, recovery from inactivation, and intracellular regulation process. These two currents were respectively designated I(Na1) and I(Na2) for l-glutamate-sensitive and -insensitive sodium currents. l-glutamate selectively reduced I(Na1) by an increase of intracellular cAMP level. Using different activators and/or inhibitors of G proteins and cAMP/PKA cascade, together with St-Ht31 (an inhibitor of PKA binding to AKAP) and AKAP-79 antibodies, we established that mGluRIII was linked to I(Na1) by a Gi/o and a suspected Gs protein. According to the activated signaling pathway, l-glutamate elevated the cAMP level, which thereby activated cytosolic PKA and released PKA bound to AKAP. As expected from both biophysical and pharmacological studies, we showed that, through an inhibition of I(Na1), l-glutamate increased DUM neuron spontaneous electrical activity. These results indicated that such mGluRIII-activated dual processes provided a new physiological control of pacemaker neuronal firing.

How Does Calcium-Dependent Intracellular Regulation of Voltage-Dependent Sodium Current Increase the Sensitivity to the Oxadiazine Insecticide Indoxacarb Metabolite Decarbomethoxylated JW062 (DCJW) in Insect Pacemaker Neurons?

“…2, IC 50 1 ϭ 1.5 nM and IC 50 2 ϭ 2.5 M,). These first results seemed to indicate that the two sodium channels, expressed in DUM neuron cell bodies (Lavialle- Defaix et al, 2006), could also be differentiated on the basis of their sensitivity to intracellular ATP concentration, thereby unmasking differing DCJW sensitivities. To substantiate this hypothesis, we designed additional experiments to study the effects of DCJW on INa1 and INa2 in isolation.…”

“…Figure 1C shows the comparative semilogarithmic curves of INa1 and INa2 inhibition induced by bath application of DCJW. The inhibitory effect of DCJW on INa2 was obtained after bath application of 10 M L-glutamate, which is known to produce full inhibition of INa1 (Lavialle- Defaix et al, 2006). The effect of DCJW on INa1 was obtained by subtraction of Lglutamate-resistant INa from the corresponding global sodium current (Fig.…”

“…Solutions and Chemicals. The superfusing solution and the internal pipette solution used to record inward sodium currents were designed to eliminate any interference from calcium and potassium currents as already described elsewhere (Lapied et al, 1990;Lavialle-Defaix et al, 2006). The extracellular solution contained 80 mM NaCl, 100 mM TEA-Cl, 3.1 mM KCl, 2 mM CaCl 2 (except when otherwise stated), 7 mM MgCl 2 , 1 mM CdCl 2 , 5 mM 4-AP, and 10 mM HEPES buffer; pH was adjusted to 7.4 with NaOH.…”

“…Consequently, in the present study, we investigated the influence of sodium-channel intracellular regulation on the efficiency of DCJW using a whole-cell patch-clamp technique adapted on short-term cultured insect pacemaker neurons (Grolleau and Lapied, 2000;Wicher et al, 2001). In previous studies, we have demonstrated the coexistence of two inward voltage-dependent sodium currents, named INa1 and INa2, in the same insect pacemaker neuron (Lavialle- Defaix et al, 2006). Here, we report that these two currents differ in their sensitivity to DCJW, with INa2 being 1000-fold less sensitive than INa1.…”

Metabotropic Glutamate Receptor Subtype 8 in the Amygdala Modulates Thermal Threshold, Neurotransmitter Release, and Rostral Ventromedial Medulla Cell Activity in Inflammatory Pain

“…( S)-3,4-DCPG and MSOP were purchased from Tocris Bioscience. ( S)-3,4-DCPG and MSOP were dissolved in ACSF (vehicle) on the day of the experiment at a concentration 100 times that predicted to be needed in the tissue based on data in the literature (Thomas et al, 2001;Lavialle-Defaix et al, 2006;Marabese et al, 2007b;Palazzo et al, 2008) because of the concentration gradient across the dialysis membrane Fu and Neugebauer, 2008). The numbers given in this article refer to the drug concentrations in the microdialysis fiber.…”