Add time:08/02/2019         Source:sciencedirect.com

Targeting signalling molecules to ion channels can expedite regulation and assure the proper transition of changes to excitability. In the bag cell neurons of Aplysia, single-channel studies of excised patches have revealed that protein kinase C (PKC) gates a non-selective cation channel through a close, physical association. This channel drives a prolonged afterdischarge and concomitant neuropeptide secretion to provoke reproductive behaviour. However, it is not clear if PKC alters cation channel function and/or the membrane potential at the whole-cell level. Afterdischarge-like depolarizations can be evoked in cultured bag cell neurons by bath-application of Conus textile venom (CtVm), which triggers the cation channel through an apparent intracellular pathway. The present study shows that the CtVm-induced depolarization was reduced by nearly 50% compared to control following dialysis with the G-protein blocker, guanosine-5′-O-2-thiodiphosphate (GDP-β-S), or treatment with either the phospholipase C inhibitor, 1-[6-[[(17β)-3-Methoxyestra-1,3,5(10)-trien-17-yl]amino]hexyl]-1H-pyrrole-2,5-dione (U-73122), or the PKC inhibitor, sphinganine. Neurons exposed to the PKC activator, phorbol 12-myristate 13-acetate (PMA), displayed depolarization with accompanying spiking, and were found to be far more responsive to depolarizing current injection versus control. Immunocytochemical staining for the two typical Aplysia PKC isoforms, Apl I and Apl II, revealed that both kinases were present in unstimulated cultured bag cell neurons. However, in CtVm-treated neurons, the staining intensity for PKC Apl I increased, peaking at 10 min post-application. Conversely, the intensity of PKC Apl II staining decreased over the duration of CtVm exposure. Our results suggest that the CtVm-induced depolarization involves PKC activation, and is consistent with prior work showing PKC closely-associating with the cation channel to produce the depolarization necessary for the afterdischarge and species propagation.

▶Conus textile venom depolarized cultured bag cell neurons. ▶G-protein, PLC, or PKC inhibitors blocked the venom-induced depolarization. ▶Venom increased PKC Apl I immunostaining and decreased Apl II immunostaining.

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