Abstract
The ClC protein family includes voltage-gated chloride channels and chloride/proton exchangers. In eukaryotes, ClC proteins regulate membrane potential of excitable cells, contribute to epithelial transport, and aid in lysosomal acidification. Although structure/function studies of ClC proteins have been aided greatly by the available crystal structures of a bacterial ClC chloride/proton exchanger, the availability of useful pharmacological tools, such as peptide toxin inhibitors, has lagged far behind that of their cation channel counterparts. Here we report the isolation, from Leiurus quinquestriatus hebraeus venom, of a peptide toxin inhibitor of the ClC-2 chloride channel. This toxin, GaTx2, inhibits ClC-2 channels with a voltage-dependent apparent K(D) of approximately 20 pm, making it the highest affinity inhibitor of any chloride channel. GaTx2 slows ClC-2 activation by increasing the latency to first opening by nearly 8-fold but is unable to inhibit open channels, suggesting that this toxin inhibits channel activation gating. Finally, GaTx2 specifically inhibits ClC-2 channels, showing no inhibitory effect on a battery of other major classes of chloride channels and voltage-gated potassium channels. GaTx2 is the first peptide toxin inhibitor of any ClC protein. The high affinity and specificity displayed by this toxin will make it a very powerful pharmacological tool to probe ClC-2 structure/function.
Highlights
ClC proteins form a family of voltage-gated ClϪ channels and ClϪ/Hϩ exchangers that are found in animals, plants, and bacteria [1]
The regions of the ClC protein involved in slow gating are still unknown, despite the availability of the bacterial ClC crystal structure
We recently showed that venom from the scorpion Leiurus quinquestriatus hebraeus contains a peptide component that inhibits the ClC-2 chloride channel [18]
Summary
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. Venom Preparation and Toxin Purification—Venom from L. quinquestriatus hebraeus was obtained from Latoxan (France) and prepared as described in the supplemental materials or Ref. 18. Subsequent fractions were resuspended in ND96 bath solution for the electrophysiological bio-assay (see the supplemental materials). The data were acquired at 2 kHz and filtered at a corner frequency of 500 Hz. 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). To prevent loss of material, all of the solutions containing toxin were prepared immediately prior to experiments from a 20 M stock. The differences were determined to be significant when p Ͻ 0.05, using paired and unpaired Student’s t tests
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
