Internalization of Voltage-Dependent Sodium Channels in Fetal Rat Brain Neurons: A Study of the Regulation of Endocytosis

Abstract: In fetal rat brain neurons, activation of voltage-dependent Na⁺ channels induced their own internalization, probably triggered by an increase in intracellular Na⁺ level. To investigate the role of phosphorylation in internalization, neurons were exposed to either activators or inhibitors of cyclic AMP- and cyclic GMP-dependent protein kinases, protein kinase C, and tyrosine kinase. None of the tested compounds mimicked or inhibited the effect of Na⁺ channel activation. An increase in intracellular Ca²⁺ concentration induced either by thapsigargin, a Ca²⁺-ATPase blocker, or by A23187, a Ca²⁺ ionophore, was unable to provoke Na⁺ channel internalization. However, Ca²⁺ seems to be necessary because both neurotoxin- and amphotericin B-induced Na⁺ channel internalizations were partially inhibited by BAPTA-AM. The selective inhibitor of Ca²⁺/calmodulin-dependent protein kinase II, KN-62, caused a dose-dependent inhibition of neurotoxin-induced internalization due to a blockade of channel activity but did not prevent amphotericin B-induced internalization. The rate of increase in Na⁺ channel density at the neuronal cell surface was similar before and after channel internalization, suggesting that recycling of internalized Na⁺ channels back to the cell surface was almost negligible. Pretreatment of the cells with an acidotropic agent such as chloroquine prevented Na⁺ channel internalization, indicating that an acidic endosomal/lysosomal compartment is involved in Na⁺ channel internalization in neurons.