Among ionic currents underlying neuronal pacemaker activity, low-threshold-activated calcium currents contribute to setting the threshold for spike firing. In the insect central nervous system, dorsal unpaired median (DUM) neurons are capable of generating spontaneous electrical activity. It has previously been shown that two distinct (transient and maintained) low-voltage-activated (LVA) calcium currents are responsible for the generation of the pacemaker potential. Whole-cell recordings in voltage- and current-clamp mode were obtained from short-term cultured DUM neurons. Using 100 mM sodium and 2 mM calcium as charge carrier in the external solution as well as conditions that eliminate calcium currents (0.5 mM CdCl2), voltage-clamp experiments showed that a hitherto unanticipated LVA maintained inward current, activated at around -60 mV, was present. The current amplitude was strongly dependent on internal ATP concentration. Sodium-free solution reduced by 80% the current amplitude. Increasing (5 mM) or decreasing (calcium-free) external calcium concentrations enhanced or reduced, respectively, the maximum conductance without any effect on the voltage dependence. This novel ion channel was permeable to barium but manipulating internal or external magnesium concentrations was without effect on current amplitude or reversal potential. Based on IC50 values, the maintained current was 50-fold less sensitive to TTX than the classical transient voltage-dependent sodium current. Furthermore, it was insensitive to ethosuximide and halothane. Voltage-dependent inactivation analysis revealed an unexpected calcium-sensitive process that involved calcineurin. From these results it appears that, besides the two LVA calcium currents previously described, another LVA maintained inward current permeable to both sodium and calcium was also involved in the generation of the predepolarization. Based on these findings, we propose that a novel calcium-dependent mechanism is involved in the regulation of the pacemaker activity.
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.
Identification of the different intracellular pathways that control phosphorylation/dephosphorylation process of ionic channels represents an exciting alternative approach for studying the ionic mechanisms underlying neuronal pacemaker activity. In the central nervous system of the cockroach Periplaneta americana, octopaminergic neurons, called dorsal unpaired median (DUM; DUM neurons), generate spontaneous repetitive action potentials. Short-term cultured adult DUM neurons isolated from the terminal abdominal ganglion (TAG) of the nerve cord were used to study the regulation of a tetrodotoxin-sensitive low-voltage-activated (LVA) channel permeable to sodium and calcium (Na/Ca), under whole cell voltage- and current-clamp conditions. A bell-shaped curve illustrating the regulation of the amplitude of the maintained current vs. [ATP]i was observed. This suggested the existence of phosphorylation mechanisms. The protein kinase A (PKA) inhibitor, H89 and elevating [cyclic adenosine 3′, 5′ monophosphate, cAMP]i, increased and decreased the current amplitude, respectively. This indicated a regulation of the current via a cAMP/PKA cascade. Furthermore, intracellular application of PP2B inhibitors, cyclosporine A, FK506 and PP1/2A inhibitor, okadaic acid decreased the current amplitude. From these results and because octopamine (OA) regulates DUM neuron electrical activity via an elevation of [cAMP]i, we wanted to know if, like in vertebrate dopaminergic neurons, OA receptor (OAR) stimulation could indirectly affect the current via PKA-mediated phosphorylation of Dopamine- and cAMP-regulated Phosphoprotein-32 (DARPP-32) known to inhibit PP1/2A. Experiments were performed using intracellular application of phospho-DARPP-32 and non-phospho-DARPP-32. Phospho-DARPP-32 strongly reduced the current amplitude whereas non-phospho-DARPP-32 did not affect the current. All together, these results confirm that DARPP-32-mediated inhibition of PP1/2A regulates the maintained sodium/calcium current, which contributes to the development of the pre-depolarizing phase of the DUM neuron pacemaker activity.
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