Activity-Induced Internalization and Rapid Degradation of Sodium Channels in Cultured Fetal Neurons

• Published in: The Journal of cell biology Vol. 134; no. 2; pp. 499 - 509
• Main Authors: Paillart, Christophe, Boudier, Jean-Louis, Boudier, Jeanne-Andrée, Rochat, Hervé, Couraud, François, Dargent, Bénédicte
• Format: Journal Article
• Language: English
• Published: United States Rockefeller University Press 01.07.1996; The Rockefeller University Press
• Subjects: Amphotericin B - pharmacology; Animals; Brain; Brain - cytology; Brain - embryology; Cell membranes; Cells, Cultured; Circles; Down regulation; Endocytosis; Internalization; Iodine Radioisotopes; Leupeptins - pharmacology; Molecular biology; Neurons; Neurons - cytology; Neurons - drug effects; Neurons - metabolism; Neurons - ultrastructure; Radioactive decay; Rats; Rats, Wistar; Scorpion Venoms - metabolism; Scorpiones; Scorpions - metabolism; Sodium; Sodium channels; Sodium Channels - drug effects; Sodium Channels - metabolism; Time; Toxins; Veratridine - pharmacology
• ISSN: 0021-9525; 1540-8140
• DOI: 10.1083/jcb.134.2.499

A regulatory mechanism for neuronal excitability consists in controlling sodium channel density at the plasma membrane. In cultured fetal neurons, activation of sodium channels by neurotoxins, e.g., veratridine and α-scorpion toxin (α-ScTx) that enhance the channel open state probability induced a rapid down-regulation of surface channels. Evidence that the initial step of activity-induced sodium channel down-regulation is mediated by internalization was provided by using125I-α-ScTx as both a channel probe and activator. After its binding to surface channels, the distribution of125I-α-ScTx into five subcellular compartments was quantitatively analyzed by EM autoradiography.125I-α-ScTx was found to accumulate in tubulovesicular endosomes and disappear from the cell surface in a time-dependent manner. This specific distribution was prevented by addition of tetrodotoxin (TTX), a channel blocker. By using a photoreactive derivative to covalently label sodium channels at the surface of cultured neurons, we further demonstrated that they are degraded after veratridine-induced internalization. A time-dependent decrease in the amount of labeled sodium channel α subunit was observed after veratridine treatment. After 120 min of incubation, half of the α subunits were cleaved. This degradation was prevented totally by TTX addition and was accompanied by the appearance of an increasing amount of a 90-kD major proteolytic fragment that was already detected after 45-60 min of veratridine treatment. Exposure of the photoaffinity-labeled cells to amphotericin B, a sodium ionophore, gave similar results. In this case, degradation was prevented when Na+ions were substituted by choline ions and not blocked by TTX. After veratridine- or amphotericin B-induced internalization of sodium channels, breakdown of the labeled α subunit was inhibited by leupeptin, while internalization was almost unaffected. Thus, cultured fetal neurons are capable of adjusting sodium channel density by an activity-dependent endocytotic process that is triggered by Na+influx.