Melatonin-mediated inhibition of Purkinje neuron P-type Ca2+ channels in vitro induces neuronal hyperexcitability through the phosphatidylinositol 3-kinase-dependent protein kinase C delta pathway

摘要

Although melatonin receptors are widely expressed in the mammalian central nervous system and peripheral tissues, there are limited data regarding the functions of melatonin in cerebellar Purkinje cells. Here, we identified a novel functional role of melatonin in modulating P-type Ca2+ channels and action-potential firing in rat Purkinje neurons. Melatonin at 0.1m reversibly decreased peak currents (I-Ba) by 32.9%. This effect was melatonin receptor 1 (MTR1) dependent and was associated with a hyperpolarizing shift in the voltage dependence of inactivation. Pertussis toxin pretreatment, intracellular application of QEHA peptide, and a selective antibody raised against the G subunit prevented the inhibitory effects of melatonin. Pretreatment with phosphatidylinositol 3-kinase (PI3K) inhibitors abolished the melatonin-induced decrease in I-Ba. Surprisingly, melatonin responses were not regulated by Akt, a common downstream target of PI3K. Melatonin treatment significantly increased protein kinase C (PKC) activity 2.1-fold. Antagonists of PKC, but not of protein kinase A, abolished the melatonin-induced decrease in I-Ba. Melatonin application increased the membrane abundance of PKC, and PKC inhibition (either pharmacologically or genetically) abolished the melatonin-induced I-Ba response. Functionally, melatonin increased spontaneous action-potential firing by 53.0%; knockdown of MTR1 and blockade of P-type channels abolished this effect. Thus, our results suggest that melatonin inhibits P-type channels through MTR1 activation, which is coupled sequentially to the subunits of G(i/o)-protein and to downstream PI3K-dependent PKC signaling. This likely contributes to its physiological functions, including spontaneous firing of cerebellar Purkinje neurons.