Substitutions in the domain III voltage-sensing module enhance the sensitivity of an insect sodium channel to a scorpion beta-toxin

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.     

Structural basis of neutralization of the major toxic component from the scorpion Centruroides noxius Hoffmann by a human-derived single-chain antibody fragment

It has previously been reported that several single-chain antibody fragments of human origin (scFv) neutralize the effects of two different scorpion venoms through interactions with the primary toxins of Centruroides noxius Hoffmann (Cn2) and Centruroides suffusus suffusus (Css2). Here we present the crystal structure of the complex formed between one scFv (9004G) and the Cn2 toxin, determined in two crystal forms at 2.5 and 1.9 Å resolution. A 15-residue span of the toxin is recognized by the antibody through a cleft formed by residues from five of the complementarity-determining regions of the scFv. Analysis of the interface of the complex reveals three features. First, the epitope of toxin Cn2 overlaps with essential residues for the binding of β-toxins to its Na⁺ channel receptor site. Second, the putative recognition of Css2 involves mainly residues that are present in both Cn2 and Css2 toxins. Finally, the effect on the increase of affinity of previously reported key residues during the maturation process of different scFvs can be inferred from the structure. Taken together, these results provide the structural basis that explain the mechanism of the 9004G neutralizing activity and give insight into the process of directed evolution that gave rise to this family of neutralizing scFvs.     

Arrangement and mobility of the voltage sensor domain in prokaryotic voltage-gated sodium channels

Prokaryotic voltage-gated sodium channels (Na(V)s) form homotetramers with each subunit contributing six transmembrane α-helices (S1-S6). Helices S5 and S6 form the ion-conducting pore, and helices S1-S4 function as the voltage sensor with helix S4 thought to be the essential element for voltage-dependent activation. Although the crystal structures have provided insight into voltage-gated K channels (K(V)s), revealing a characteristic domain arrangement in which the voltage sensor domain of one subunit is close to the pore domain of an adjacent subunit in the tetramer, the structural and functional information on Na(V)s remains limited. Here, we show that the domain arrangement in NaChBac, a firstly cloned prokaryotic Na(V), is similar to that in K(V)s. Cysteine substitutions of three residues in helix S4, Q107C, T110C, and R113C, effectively induced intersubunit disulfide bond formation with a cysteine introduced in helix S5, M164C, of the adjacent subunit. In addition, substituting two acidic residues with lysine, E43K and D60K, shifted the activation of the channel to more positive membrane potentials and consistently shifted the preferentially formed disulfide bond from T110C/M164C to Q107C/M164C. Because Gln-107 is located closer to the extracellular side of helix S4 than Thr-110, this finding suggests that the functional shift in the voltage dependence of activation is related to a restriction of the position of helix S4 in the lipid bilayer. The domain arrangement and vertical mobility of helix S4 in NaChBac indicate that the structure and the mechanism of voltage-dependent activation in prokaryotic Na(V)s are similar to those in canonical K(V)s.     

A molecular switch between the outer and the inner vestibules of the voltage-gated Na+ channel

Voltage-gated ion channels are transmembrane proteins that undergo complex conformational changes during their gating transitions. Both functional and structural data from K(+) channels suggest that extracellular and intracellular parts of the pore communicate with each other via a trajectory of interacting amino acids. No crystal structures are available for voltage-gated Na(+) channels, but functional data suggest a similar intramolecular communication involving the inner and outer vestibules. However, the mechanism of such communication is unknown. Here, we report that amino acid Ile-1575 in the middle of transmembrane segment 6 of domain IV (DIV-S6) in the adult rat skeletal muscle isoform of the voltage-gated sodium channel (rNa(V)1.4) may act as molecular switch allowing for interaction between outer and inner vestibules. Cysteine scanning mutagenesis of the internal part of DIV-S6 revealed that only mutations at site 1575 rescued the channel from a unique kinetic state ("ultra-slow inactivation," I(US)) produced by the mutation K1237E in the selectivity filter. A similar effect was seen with I1575A. Previously, we reported that conformational changes of both the internal and the external vestibule are involved in the generation of I(US). The fact that mutations at site 1575 modulate I(US) produced by K1237E strongly suggests an interaction between these sites. Our data confirm a previously published molecular model in which Ile-1575 of DIV-S6 is in close proximity to Lys-1237 of the selectivity filter. Furthermore, these functional data define the position of the selectivity filter relative to the adjacent DIV-S6 segment within the ionic permeation pathway.     

Structural implications on the interaction of scorpion alpha-like toxins with the sodium channel receptor site inferred from toxin iodination and pH-dependent binding

The alpha-like toxin from the venom of the scorpion Leiurus quinquestriatus hebraeus (Lqh III) binds with high affinity to receptor site 3 on insect sodium channels but does not bind to rat brain synaptosomes. The binding affinity of Lqh III to cockroach neuronal membranes was fivefold higher at pH 6.5 than at pH 7.5. This correlated with an increase in the electropositive charge on the toxin surface resulting from protonation of its four histidines. Radioiodination of Tyr(14) of Lqh III abolished its binding to locust but not cockroach sodium channels, whereas the noniodinated toxin bound equally well to both neuronal preparations. Radioiodination of Tyr(10) or Tyr(21) of the structurally similar alpha-toxin from L. quinquestriatus hebraeus (LqhalphaIT), as well as their substitution by phenylalanine, had only minor effects on binding to cockroach neuronal membranes. However, substitution of Tyr(21), but not Tyr(14), by leucine decreased the binding affinity of LqhalphaIT approximately 87-fold. Thus, Tyr(14) is involved in the bioactivity of Lqh III to locust receptor site 3 and is not crucial for the binding of LqhalphaIT to this site. In turn, the aromatic ring of Tyr(21) takes part in the bioactivity of LqhalphaIT to insects. These results highlight subtle architectural variations between locust and cockroach receptor site 3, in addition to previous results demonstrating the competence of Lqh III to differentiate between insect and mammalian sodium channel subtypes.