ACTIVITY-INDUCED INTERNALIZATION AND RAPID DEGRADATION OF SODIUM-CHANNELS IN CULTURED FETAL NEURONS

A regulatory mechanism for neuronal excitability consists in controlli ng sodium channel density at the plasma membrane. In cultured fetal ne urons, activation of sodium channels by neurotoxins, e.g., veratridine and alpha-scorpion toxin (alpha-ScTx) that enhance the channel open s tate probability induced a rapid downregulation of surface channels. E vidence that the initial step of activity-induced sodium channel down- regulation is mediated by internalization was provided by using I-125- alpha-ScTX as both a channel probe and activator. After its binding to surface channels, the distribution of I-125-alpha-ScTX into five subc ellular compartments was quantitatively analyzed by EM autoradiography . I-125-alpha- ScTx was found to accumulate in tubulovesicular endosom es and disappear from the cell surface in a time-dependent manner. Thi s specific distribution was prevented by addition of tetrodotoxin (TTX ), a channel blocker. By using a photoreactive derivative to covalentl y label sodium channels at the surface of cultured neurons, we further demonstrated that they are degraded after veratridine-induced interna lization. A time-dependent decrease in the amount of labeled sodium ch annel alpha subunit was observed after veratridine treatment. After 12 0 min of incubation, half of the alpha subunits were cleaved. This deg radation was prevented totally by TTX addition and was accompanied by the appearance of an increasing amount of a 90-kD major proteolytic fr agment that was already detected after 45-60 min of veratridine treatm ent. 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 block ed by TTX. After veratridine- or amphotericin B-induced internalizatio n of sodium channels, breakdown of the labeled alpha subunit was inhib ited 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.