ω-conotoxin exerts functionally distinct low and high affinity effects in the neuronal cell line NG108-15

The Ca²⁺ channel blockade produced by ω-conotoxin GVIA (ω-CgTx) was studied in single, forskolin-differentiated, NG108-15 cells, using dual-emission microfluorimetry and the whole-cell patch-clamp technique. Whole-cell currents through Ca²⁺ channels were measured with 5 mM Ba²⁺ as the charge carrier. Application of 1 μM nitrendipine inhibited by 90% the currents evoked by stepping from -30 mV to 0 mV. ω-CgTx (1 μM) inhibited these currents by 28%. These data suggest the possibility that NG108-15 cells express two types of dihydropyridinesensitive Ca²⁺ channel, one sensitive and the other insensitive to blockade by ω-CgTx. The nature of the Ca²⁺ channel blockade produced by these agents was studied further, using depolarization-induced intracellular free Ca²⁺ concentration ([Ca²⁺]_i) transients recorded with the Ca²⁺ indicator indo-1 and a dual-emission microfluorimeter. A 30-sec superfusion with 50 mM K⁺ increased the [Ca²⁺J_i from a basal level of 142 ± 10 nM to a peak level of 1655 ± 287 nM. This [Ca²⁺]_i transient was blocked completely and reversibly by nitrendipine, in a concentration-dependent manner (IC₅₀ = 1.9nM). In contrast, ω-CgTx produced a maximal inhibition of the depolarization-induced rise in [Ca²⁺]_i of only 52% in the presence of physiological concentrations of divalent metals. The block was irreversible. This inhibition was concentration dependent until the point of maximal inhibition, at which point the channel block reversed in a graded manner. This entire U-shaped dose-response curve could be shifted in a parallel fashion by modulation of the extracellular divalent metal concentration, without changes in the maximal inhibition. Repeated applications of or prolonged incubations with ω-CgTx failed to increase the maximal block. Treatment with a high (1 μM) concentration of ω-CgTx, which produced a modest (10%) inhibition of Ca²⁺ influx, protected the cell from a second exposure to a normally effective concentration of ω-CgTx (10 nM). Depolarization-induced [Ca²⁺] transients in cells treated with 10 nM toxin were inhibited by 45%, and this inhibition could not be reversed by subsequent exposure to a high concentration of ω-CgTx. We conclude that there are two ω-CgTx binding sites on these cells, one to which ω-CgTx binds with high affinity, producing an irreversible Ca²⁺ channel blockade, and a second to which ω-CgTx binds with lower affinity. Binding to this second site is irreversible and does not block the channel but does prevent access to the high affinity site. These data suggest caution in the use of ω-CgTx as a tool to distinguish Ca²⁺ channel subtypes.