Inhibition of ClC-2 Chloride Channels by a Peptide Component or Components of Scorpion Venom

Abstract

ClC chloride channels play essential roles in membrane excitability and maintenance of osmotic balance. Despite the recent crystallization of two bacterial ClC-like proteins, the gating mechanism for these channels remains unclear. In this study we tested scorpion venom for the presence of novel peptide inhibitors of ClC channels, which might be useful tools for dissecting the mechanisms underlying ClC channel gating. Recently, it has been shown that a peptide component of venom from the scorpion L. quinquestriatus hebraeus inhibits the CFTR chloride channel from the intracellular side. Using two-electrode voltage clamp we studied the effect of scorpion venom on ClC-0, -1, and -2, and found both dose- and voltage-dependent inhibition only of ClC-2. Comparison of voltage-dependence of inhibition by venom to that of known pore blockers revealed opposite voltage dependencies, suggesting different mechanisms of inhibition. Kinetic data show that venom induced slower activation kinetics compared to pre-venom records, suggesting that the active component(s) of venom may function as a gating modifier at ClC-2. Trypsinization abolished the inhibitory activity of venom, suggesting that the component(s) of scorpion venom that inhibits ClC-2 is a peptide.

Isolation and Characterization of a High Affinity Peptide Inhibitor of ClC-2 Chloride Channels

“…Isolation and Proteomic Characterization of the Active ToxinWe recently showed that a peptide component of scorpion venom is capable of specifically inhibiting the ClC-2 chloride channel (18). To isolate the active species from venom, we used RP-HPLC to separate the components of Lqh pf-venom, which contains only components smaller than 10 kDa.…”

“…However, no peptide toxins have been isolated that inhibit a ClC channel, and only one toxin has been isolated that inhibits any Cl Ϫ channel of known molecular identity (17). We recently showed that venom from the scorpion Leiurus quinquestriatus hebraeus contains a peptide component that inhibits the ClC-2 chloride channel (18). Here, we report the isolation of this peptide toxin, its proteomic properties, and primary characteristics of the biophysical mechanism of inhibition.…”

“…Bath solutions and voltage protocols for TEVC experiments with ClC channels were as described in the supplemental materials or Ref. 18. Inside-out multi-channel patch, inside-out and outside-out macropatch, and singlechannel patch clamp experiments were performed as described previously (17,18), using an Axopatch 200B amplifier (Axon).…”

“…Oocyte and cRNA Preparation-Xenopus oocytes were isolated as described previously (18) and incubated at 18°C in a modified Liebovitz's L-15 medium (pH 7.5) with a mixture of antibiotics (gentamicin, penicillin, and streptomycin) and HEPES. The oocytes were injected with cRNAs encoding ClC-1, -2, -3, and -4; CFTR 5 ; GABA C ; Shaker B-IR; or K V 1.2.…”

“…18. Inside-out multi-channel patch, inside-out and outside-out macropatch, and singlechannel patch clamp experiments were performed as described previously (17,18), using an Axopatch 200B amplifier (Axon). For all recordings the dashed line represents the zero current level.…”