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

Roy Kahn,Izhar Karbat,Nitza Ilan,Lior Cohen,Stanislav Sokolov,William A Catterall,Dalia Gordon,Michael Gurevitz

doi:10.1074/jbc.m109.021303

Roy Kahn, Izhar Karbat + Show 6 more

Open Access

https://doi.org/10.1074/jbc.m109.021303

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Abstract

The scorpion alpha-toxin Lqh2 (from Leiurus quinquestriatus hebraeus) is active at various mammalian voltage-gated sodium channels (Na(v)s) and is inactive at insect Na(v)s. To resolve the molecular basis of this preference we used the following strategy: 1) Lqh2 was expressed in recombinant form and key residues important for activity at the rat brain channel rNa(v)1.2a were identified by mutagenesis. These residues form a bipartite functional surface made of a conserved "core domain" (residues of the loops connecting the secondary structure elements of the molecule core), and a variable "NC domain" (five-residue turn and the C-tail) as was reported for other scorpion alpha-toxins. 2) The functional role of the two domains was validated by their stepwise construction on the similar scaffold of the anti-insect toxin LqhalphaIT. Analysis of the activity of the intermediate constructs highlighted the critical role of Phe(15) of the core domain in toxin potency at rNa(v)1.2a, and has suggested that the shape of the NC-domain is important for toxin efficacy. 3) Based on these findings and by comparison with other scorpion alpha-toxins we were able to eliminate the activity of Lqh2 at rNa(v)1.4 (skeletal muscle), hNa(v)1.5 (cardiac), and rNa(v)1.6 channels, with no hindrance of its activity at Na(v)1.1-1.3. These results suggest that by employing a similar approach the design of further target-selective sodium channel modifiers is imminent.

Highlights

The pivotal role of voltage-gated sodium channels (Navs)4 in excitability mark them as major targets for a large variety of toxins that bind at distinct receptor sites and modify their gating [1]
At least nine genes encode a variety of Nav subtypes [1, 3], whose expression varies greatly in different tissues (Nav1.1–1.3 mainly in the central nervous system; Nav1.6 in both central and peripheral neurons; Nav1.7 in the peripheral nervous system; Nav1.8 and Nav1.9 in sensory neurons; Nav1.4 and Nav1.5 in skeletal and cardiac muscles, respectively)
Based on the findings that site-3 toxins eliminate a gating charge component associated with the movement of D4/S4 [15, 16], and that this segment plays a critical role in coupling channel inactivation to activation [17], scorpion ␣-toxins were postulated to inhibit channel inactivation by hindering the outward movement of this segment during depolarization [9]

Summary

Introduction

The pivotal role of voltage-gated sodium channels (Navs)4 in excitability mark them as major targets for a large variety of toxins that bind at distinct receptor sites and modify their gating [1]. The correlation of this structural difference with toxin preference for Nav subtypes was corroborated by constructing the bioactive surface of Lqh␣IT on the scaffold of the anti-mammalian ␣-toxin Aah2 ending up with a chimera (Aah2Lqh␣IT(face)) active on insects, whose NC domain is in the protruding conformation [21].

Results

Conclusion