Chlorotoxin does not inhibit volume-regulated, calcium-activated and cyclic AMP-activated chloride channels

1 It was the aim of this study to look for a high-affinity and selective po lypeptide toxin, which could serve as a probe for the volume-regulated anio n channel (VRAC) or the calcium-activated chloride channel (CaCC). We have partially purified chlorotoxin, including new and homologous short chain in sectotoxins, from the crude venom of Leiurus quinquestriatus quinquestriatu s (Lqq) by means of gel filtration chromatography. Material eluting between 280 and 420 min, corresponding to fractions 15-21, was lyophilized and tes ted on VRAC and CaCC, using the whole-cell patch clamp technique. We have a lso tested the commercially available chlorotoxin on VRAC, CaCC, the cystic fibrosis transmembrane conductance regulator (CFTR) and on the glioma spec ific chloride channel (GCC). 2 VRAC and the correspondent current, I-Cl.swell, was activated in Cultured Pulmonary Artery Endothelial (CPAE) cells by a 25% hypotonic solution. Nei ther of the fractions 16-21 significantly inhibited I-Cl,I-swell (n = 4-5). Ca2+-activated Cl- currents, I-Cl,I-Ca, activated by loading T84 cells via the patch pipette with 1 mu M free Ca2+, were not inhibited by any of the tested fractions (15-21), (n = 2-5). 3 Chlorotoxin (625 nM) did neither effect I-Cl,I-swell nor I-Cl,I-Ca (n = 4 -5). The CFTR channel, transiently transfected in COS cells and activated b y a cocktail containing IBMX and forskolin, was not affected by 1.2 mu M ch lorotoxin (n = 5). In addition, it did not affect currents through GCC. 4 We conclude that submicromolar concentrations of chlorotoxin do not block volume-regulated, Ca2+-activated and CFTR chloride channels and that it ca n not be classified as a general chloride channel toxin.