Abstract
Scorpion β-toxins bind to the extracellular regions of the voltage-sensing module of domain II and to the pore module of domain III in voltage-gated sodium channels and enhance channel activation by trapping and stabilizing the voltage sensor of domain II in its activated state. We investigated the interaction of a highly potent insect-selective scorpion depressant β-toxin, Lqh-dprIT(3), from Leiurus quinquestriatus hebraeus with insect sodium channels from Blattella germanica (BgNa(v)). Like other scorpion β-toxins, Lqh-dprIT(3) shifts the voltage dependence of activation of BgNa(v) channels expressed in Xenopus oocytes to more negative membrane potentials but only after strong depolarizing prepulses. Notably, among 10 BgNa(v) splice variants tested for their sensitivity to the toxin, only BgNa(v)1-1 was hypersensitive due to an L1285P substitution in IIIS1 resulting from a U-to-C RNA-editing event. Furthermore, charge reversal of a negatively charged residue (E1290K) at the extracellular end of IIIS1 and the two innermost positively charged residues (R4E and R5E) in IIIS4 also increased the channel sensitivity to Lqh-dprIT(3). Besides enhancement of toxin sensitivity, the R4E substitution caused an additional 20-mV negative shift in the voltage dependence of activation of toxin-modified channels, inducing a unique toxin-modified state. Our findings provide the first direct evidence for the involvement of the domain III voltage-sensing module in the action of scorpion β-toxins. This hypersensitivity most likely reflects an increase in IIS4 trapping via allosteric mechanisms, suggesting coupling between the voltage sensors in neighboring domains during channel activation.
Highlights
Voltage-gated sodium (Nav) channels are essential for the initiation and propagation of action potentials in most excitable cells
The toxins that affect Nav channels are divided into ␣- and -classes according to their mode of action and binding properties [14, 15]. ␣-Toxins inhibit channel fast inactivation in a voltage-dependent manner upon binding at receptor site 3, assigned mainly to domains I and IV. -Toxins interact with receptor site 4, assigned to the voltagesensing module of domain II and the pore module of domain III, and shift the voltage dependence of activation to more negative membrane potentials (11, 16 –19)
Their analysis revealed that three amino acid residues in the C-terminal pore loop (SS2–S6) of domain III determine the Tz1 preference for the skeletal muscle Nav channel [18]
Summary
Toxin Production and Functional Analysis—Of eight LqhdprIT3 variants produced by the scorpion Leiurus quinquestriatus hebraeus, the highly potent variant c, hereafter referred to as Lqh-dprIT3, was produced in recombinant form and analyzed as described previously [26]. Expression of BgNav Channels in Xenopus Oocytes—The procedures for oocyte preparation and cRNA injection are identical to those described previously [27]. Electrophysiological Recording and Analysis—The voltage dependence of activation and inactivation was measured using the two-electrode voltage clamp technique. The voltage dependence of sodium channel conductance (G) was calculated by measuring the peak current at test potentials ranging from Ϫ80 to ϩ65 mV in 5-mV increments and divided by (V Ϫ Vrev), where V is the test potential and Vrev is the reversal potential for sodium ions. The percentage of channel modification by Lqh-dprIT3 was determined by the percentage of channels with the voltage dependence of activation shifted to negative membrane potentials, which was derived from double Boltzmann fits of the conductance-voltage relationships. Statistical significance was determined by one-way analysis of variance (p Ͻ 0.05)
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