INTERNALIZATION OF VOLTAGE-DEPENDENT SODIUM-CHANNELS IN FETAL-RAT BRAIN NEURONS - A STUDY OF THE REGULATION OF ENDOCYTOSIS

In fetal rat brain neurons, activation of voltage-dependent Na+ channe ls induced their own internalization, probably triggered by an increas e in intracellular Na+ level. To investigate the role of phosphorylati on in internalization, neurons were exposed to either activators or in hibitors of cyclic AMP- and cyclic GMP-dependent protein kinases, prot ein kinase C, and tyrosine kinase. None of the tested compounds mimick ed or inhibited the effect of Na+ channel activation. An increase in i ntracellular 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 beca use both neurotoxin- and amphotericin B-induced Na+ channel internaliz ations were partially inhibited by BAPTA-AM. The selective inhibitor o f Ca2+/calmodulin-dependent protein kinase II, KN-62, caused a dose-de pendent inhibition of neurotoxin-induced internalization due to a bloc kade of channel activity but did not prevent amphotericin B-induced in ternalization. The rate of increase in Na+ channel density at the neur onal cell surface was similar before and after channel internalization , suggesting that recycling of internalized Na+ channels back to the c ell surface was almost negligible. Pretreatment of the cells with an a cidotropic agent such as chloroquine prevented Na+ channel internaliza tion, indicating that an acidic endosomal/lysosomal compartment is inv olved in Na+ channel internalization in neurons.