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

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 ϳ20 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 voltagegated 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.

Differentiation between human ClC-2 and CFTR Cl− channels with pharmacological agents

“…However, methadone had no effect on forskolin-IBMX-stimulated Cl Ϫ currents in recombinant hCFTR/HEK293 cells. Recently, the peptide toxin inhibitor GaTx2 has become available (47). It does not inhibit CFTR, but it potently inhibits ClC-2 (IC 50 ϳ20 nM).…”

“…Recently, methadone was identified to be an inhibitor of recombinant human ClC-2 (hClC-2), but not recombinant human CFTR (hCFTR), with half-maximal inhibition (IC 50 ) at 100 nM (7), suggesting that it can be used to differentially inhibit ClC-2, but not CFTR. Interestingly, recently, a peptide toxin inhibitor, GaTx2, was identified and shown to differentiate between ClC-2 and CFTR (47). It potently, but partially, inhibited ClC-2 (IC 50 ϳ20 pM) by ϳ80% and had no effect on CFTR, even at 60 nM.…”

Dual activation of CFTR and CLCN2 by lubiprostone in murine nasal epithelia

“…The availability of a more potent and specific peptide inhibitor of CLCN2 (36) may improve the ability to confidently determine CLCN2 contribution over less specific inhibitors. GaTx2 is a peptide isolated from the venom of the scorpion Leiurus quinquestriatus hebraeus (Lqh) and shows no inhibitory affect on ClC-1, ClC-3, ClC-4, CFTR, GABA c , Ca 2ϩ -dependent Cl Ϫ channels, or Shaker ShB-IR K ϩ channels (36). It inhibits CLCN2 at concentrations as low as 100 pM in vitro but shows its highest rates of inhibition at 10 nM (36).…”

“…GaTx2 is a peptide isolated from the venom of the scorpion Leiurus quinquestriatus hebraeus (Lqh) and shows no inhibitory affect on ClC-1, ClC-3, ClC-4, CFTR, GABA c , Ca 2ϩ -dependent Cl Ϫ channels, or Shaker ShB-IR K ϩ channels (36). It inhibits CLCN2 at concentrations as low as 100 pM in vitro but shows its highest rates of inhibition at 10 nM (36). As expected, we found that in vivo somewhat higher concentrations were required to show pronounced inhibition (100 -400 nM, Figs.…”

Novel diuretic targets

“…Nevertheless, recent progress demonstrates the potential for development of novel ClC-specific compounds. Compounds specific to the ClC-2 channel and to the ClC-ec1 antiporter have recently been described (59,137). Of relevance to the kidney, Pusch and colleagues (48) have systematically characterized and developed small-molecule inhibitors and activators of the ClC-K channels.…”