Effect of Depolarization on Binding Kinetics of Scorpion α-Toxin Highlights Conformational Changes of Rat Brain Sodium Channels

Abstract

Binding of scorpion alpha-toxins to receptor site 3 on voltage-gated sodium channels inhibits sodium current inactivation and is voltage-dependent. To reveal the direct effect of depolarization, we analyzed binding kinetics of the alpha-toxin Lqh-II (from Leiurus quinquestriatus hebraeus) to rat brain synaptosomes and effects on rat brain II (rBII) channels expressed in mammalian cells. Our results indicated that the 33-fold decrease in toxin affinity for depolarized (0 mV, 90 mM [K(+)](out), K(d) = 5.85 +/- 0.5 nM) versus polarized (-55 mV, 5 mM [K(+)](out), K(d) = 0.18 +/- 0.04 nM) synaptosomes at steady state results from a 48-fold reduction in the association rate (k(on) at 5 mM [K(+)] = (12.0 +/- 4) x 10(6) M(-1) s(-1) and (0.25 +/- 0.03) x 10(6) M(-1) s(-1) at 90 mM [K(+)](out)) with nearly no change in the dissociation rate. Electrophysiological analyses of rBII channels expressed in mammalian cells revealed that approximately 75% and 40% of rBII occupied fast- and slow-inactivated states, respectively, at resting membrane potential of synaptosomes (-55 mV), and Lqh-II markedly increased the steady-state fast and slow inactivation. To mimic electrophysiological conditions we induced fast depolarization of toxin-bound synaptosomes, which generated a biphasic unbinding of Lqh-II from toxin-receptor complexes. The first fast off rate closely resembled values determined electrophysiologically for rBII in mammalian cells. The second off rate was similar to the voltage-independent steady-state value, attributed to binding to the slow-inactivated channel states. Thus, the Lqh-II voltage-dependent affinity highlights two independent mechanisms representing conformational changes of sodium channels associated with transitions among electrically visible and invisible inactivated states.

Common Features in the Functional Surface of Scorpion β-Toxins and Elements That Confer Specificity for Insect and Mammalian Voltage-gated Sodium Channels

“…Receptor site capacity for His-Css4 (B max ϭ 0.82 Ϯ 0.08 pmol/mg protein; n ϭ 4; Fig. 2) was in the same range as that obtained previously for other scorpion ␣-and ␤-toxins to rat brain synaptosomes prepared in a similar fashion (0.6 -2.4 pmol/mg) (12,13,20,31,32), suggesting that they all bound to a similar population of sodium channels.…”

“…Binding Experiments-Rat brain synaptosomes were prepared from adult albino Wistar strain (ϳ300 g, laboratory bred) as described previously (20) and kept at Ϫ70°C. Membrane protein concentration was determined by a Bio-Rad protein assay, using bovine serum albumin as a standard.…”

“…Binding buffer composition was 130 mM Choline-Cl, 1.8 mM CaCl 2 , 5.4 mM KCl, 0.8 mM MgSO 4 , 50 mM Hepes-Tris, pH 7.2, 10 mM glucose, and 2 mg/ml bovine serum albumin. Washing buffer composition was 140 mM Choline-Cl, 1.8 mM CaCl 2 , 5.4 mM KCl, 0.8 mM MgSO 4 , 50 mM Hepes-Tris, pH 7.2, and 2 mg/ml bovine serum albumin (20,22). Equilibrium competition binding assays were performed using increasing concentrations of the unlabeled toxin in the presence of a constant low concentration of 125 I-His-Css4 and analyzed by the computer program KaleidaGraph (Synergy Software) using a nonlinear Hill equation (for IC 50 determination).…”

Molecular Requirements for Recognition of Brain Voltage-gated Sodium Channels by Scorpion α-Toxins

“…Binding Experiments-Preparation of rat brain synaptosomes, membrane protein quantification, and Lqh2 radioiodination were carried out as described (29). Purification of the monoiodotoxin and determination of its concentration, as well as the composition of media used in the binding assays and termination of the reactions have been previously described (29,35).…”

“…Lqh2 is a 64-residue long toxin from L. quinquestriatus hebraeus (Israeli yellow scorpion) (27) that is almost identical in sequence (96% identity) to the most active anti-mammalian toxin, Aah2, whose structure and action are documented (18,28,29). By functional expression and mutagenesis we uncovered residues on the Lqh2 exterior that are putatively involved in bioactivity.…”