Abstract
Neurotoxin receptor site-3 at voltage-gated Na(+) channels is recognized by various peptide toxin inhibitors of channel inactivation. Despite extensive studies of the effects of these toxins, their mode of interaction with the channel remained to be described at the molecular level. To identify channel constituents that interact with the toxins, we exploited the opposing preferences of LqhαIT and Lqh2 scorpion α-toxins for insect and mammalian brain Na(+) channels. Construction of the DIV/S1-S2, DIV/S3-S4, DI/S5-SS1, and DI/SS2-S6 external loops of the rat brain rNa(v)1.2a channel (highly sensitive to Lqh2) in the background of the Drosophila DmNa(v)1 channel (highly sensitive to LqhαIT), and examination of toxin activity on the channel chimera expressed in Xenopus oocytes revealed a substantial decrease in LqhαIT effect, whereas Lqh2 was as effective as at rNa(v)1.2a. Further substitutions of individual loops and specific residues followed by examination of gain or loss in Lqh2 and LqhαIT activities highlighted the importance of DI/S5-S6 (pore module) and the C-terminal region of DIV/S3 (gating module) of rNa(v)1.2a for Lqh2 action and selectivity. In contrast, a single substitution of Glu-1613 to Asp at DIV/S3-S4 converted rNa(v)1.2a to high sensitivity toward LqhαIT. Comparison of depolarization-driven dissociation of Lqh2 and mutant derivatives off their binding site at rNa(v)1.2a mutant channels has suggested that the toxin core domain interacts with the gating module of DIV. These results constitute the first step in better understanding of the way scorpion α-toxins interact with voltage-gated Na(+)-channels at the molecular level.
Highlights
Receptor site-3 has been localized at low resolution to the extracellular loops in domains I and IV using a photoaffinitylabeled scorpion ␣-toxin (Lqq5 from Leiurus quinquestriatus quinquestriatus) and antibodies directed to specific regions of the external loops in domains I (S5–S6) and IV (S3–S4 and S5–S6) of the rat brain channel rNav1.2 [19, 20]
Based on previous reports suggesting that receptor site-3 is associated with channel external loops of domains IV and I [19, 21, 23, 24, 33], our approach to identify channel constituents that determine toxin recognition was to first uncover the extracellular loops involved with toxin selectivity and use this information to characterize toxin interaction with receptor site-3
The experimental approach in the present study was to first identify channel regions involved in toxin selectivity and dissect the relevant regions in search for specific residues associated with the receptor site
Summary
Toxin Production and Modification—Lqh was produced in Escherichia coli strain BL21 as described [29]. The voltage dependence of steady-state fast inactivation was described using a single Boltzmann distribution as shown, I/Imax ϭ ␣0 ϩ ␣1/͑1 ϩ exp͑V Ϫ Vh/k͒. The dose dependence for toxin-induced removal of fast inactivation is calculated by plotting the ratio of the steady-state current remaining 50 ms after depolarization (Iss) to the peak current (Ipeak) as a function of toxin concentration, normalized to the maximal effect set to 1, and fitted with the Hill equation, where H is the Hill coefficient, [toxin] is the toxin concentration, and a0 is the offset measured prior to toxin application. The extent of removal of fast inactivation represented by the ratio Iss/Ipeak was plotted as a function of the conditioning voltage and was fitted with the Boltzmann distribution described, IVϭ. Where V1⁄2 is the half-maximal dissociation voltage, and k1⁄2 is the corresponding slope factor
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