Scorpion ␤-toxins that affect the activation of mammalian voltage-gated sodium channels (Na v s) have been studied extensively, but little is known about their functional surface and mode of interaction with the channel receptor. To enable a molecular approach to this question, we have established a successful expression system for the anti-mammalian scorpion ␤-toxin, Css4, whose effects on rat brain Na v s have been well characterized. A recombinant toxin, His-Css4, was obtained when fused to a His tag and a thrombin cleavage site and had similar binding affinity for and effect on Na currents of rat brain sodium channels as those of the native toxin isolated from the scorpion venom. Molecular dissection of His-Css4 elucidated a functional surface of 1245 Å 2 composed of the following: 1) a cluster of residues associated with the ␣-helix, which includes a putative "hot spot" (this cluster is conserved among scorpion ␤-toxins and contains their "pharmacophore"); 2) a hydrophobic cluster associated mainly with the ␤2 and ␤3 strands, which is likely to confer the specificity for mammalian Na v s; 3) a single bioactive residue (Trp-58) in the C-tail; and 4) a negatively charged residue (Glu-15) involved in voltage sensor trapping as inferred from our ability to uncouple toxin binding from activity upon its substitution. This study expands our understanding about the mode of action of scorpion ␤-toxins and illuminates differences in the functional surfaces that may dictate their specificities for mammalian versus insect sodium channels.

No abstract

No abstract

l h e effects of anion-channel blockers on light-mediated stomatal opening, on the potassium dependence of stomatal opening, on stomatal responses to abscisic acid (ABA), and on current through slow anion channels in the plasma membrane of guard cells were investigated. The anion-channel blockers anthracene-9-carboxylic acid (9-AC) and niflumic acid blocked current through slow anion channels of Vicia faba L. guard cells. Both 9-AC and niflumic acid reversed ABA inhibition of stomatal opening in V. faba L. and Commelina communis 1. The anion-channel blocker probenecid also abolished ABA inhibition of stomatal opening in both species. Additional tests of 9-AC effects on stomatal aperture in Commelina revealed that application of this anion-channel blocker allowed wide stomatal opening under low (1 mM) KCI conditions and increased the rate of stomatal opening under both low and high (100 mM) KCI conditions. These results indicate that anion channels can function as a negative regulator of stomatal opening, presumably by allowing anion efflux and depolarization, which prohibits ion uptake in guard cells. Furthermore, 9-AC prevented ABA induction of stomatal closure. A model in which ABA activation of anion channels contributes a rate-limiting mechanism during ABA-induced stomatal closure and inhibition of stomatal opening is discussed.

The scorpion ␣-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 ␣-toxins.2) The functional role of the two domains was validated by their stepwise construction on the similar scaffold of the anti-insect toxin Lqh␣IT. 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 ␣-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.