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

Abstract

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

Figure 1

(A) Purification of ClTx and homologous short chain insectotoxins from Lqq venom. Crude venom was fractionated by FPLC gel filtration. Absorbance of the eluate was monitored at 280 nm and 4 ml fractions were collected automatically. Material eluting between 280 and 420 min, corresponding to fractions 15–21 (hollow rectangle), was lyophilized and tested on VRAC and CaCC. Based on a constructed gel filtration calibration curve, fractions 16 and 17 contain material with an average molecular weight between 3500–7000 Da. ClTx is assumed to be primarily present in fractions 16 and 17. (B) Commercially purchased chlorotoxin was checked for purity and indicated concentration by means of HPLC. A linear gradient of 0–100% 1 M NaCl was applied. The absorbance was simultaneously measured at 214, 254 and 280 nm (only shown for 214 nm).

Figure 2

(A) Time course of activation of I_Cl,swell during superfusion with hypotonic solution (HTS) (following superfusion with Krebs solution and isotonic-Cs⁺ solution (ISO)), reversible inhibition of the current by 50 μM FLX and deactivation of the current after returning to isotonic-Cs⁺ solution. The dashed line indicates zero current. Data were obtained from ramp protocols by averaging the current in small voltage windows around +100 and −150 mV. (B) Current-voltage relations obtained from voltage ramps at the times indicated in (A). (C) Time course of activation of I_Cl,swell as described in (A) and application of fraction 17 through the same delivery system as FLX. The experiment was stopped before the current deactivation was completed. (D) Current-voltage relations obtained from voltage ramps at the times indicated in (C).

Figure 3

(A) Reversible inhibition of 100 μM NA on I_Cl,Ca, activated by loading the cell with a buffered 1000 nM Ca²⁺ pipette solution (after breaking the patch at the time indicated by the arrow). The bath solution is a slightly hypertonic Krebs-Cs⁺ solution. The dashed line indicates zero current. Data were obtained from ramp protocols by averaging the current in a small voltage window around +100 and −150 mV. (B) Current-voltage relations from voltage ramps at the times indicated in (A). (C) Current traces in response to the voltage steps protocol (1 s steps, holding potential −50 mV), performed directly after the last measured voltage ramp in (A). (D) Effect of fraction 16 on I_Cl,Ca, activated as described in (A) and applied through exactly the same delivery system as NA. Because of a little time difference between breaking into the cell and the first current recording, the time course does not start from zero current as in (A). (E) Current-voltage relations from voltage ramps at the times indicated in (D). (F) Current traces in response to the voltage steps protocol (1.5 s steps, holding potential −20 mV), performed directly after the last measured voltage ramp in (D).

Figure 4

(A) Time course of activation of I_Cl,swell during superfusion with hypotonic solution (HTS) (following superfusion with Krebs solution and isotonic-Cs⁺ solution), application of 625 nM chlorotoxin and deactivation of the current after returning to isotonic-Cs⁺ solution. The dashed line indicates zero current. Data were obtained from ramp protocols by averaging the current in a small voltage window around +100 and −150 mV. (B) Current-voltage relations obtained from voltage-ramps at the times indicated in (A). (C) Time course of I_Cl,Ca, activated by loading the cell with a buffered 1000 nM Ca²⁺ pipette solution (after breaking the patch at the time indicated by the arrow) and application of 625 nM chlorotoxin. The bath solution is a slightly hypertonic Krebs-Cs⁺ solution. The dashed line indicates zero current. Data were obtained from ramp protocols by averaging the current in a small voltage window around +100 and −150 mV. (D) Current-voltage relations from voltage ramps at the times indicated in (C). (E) Effect of 1.2 μM chlorotoxin on I_Cl,cAMP currents activated with a IBMX-forskolin cocktail. Bath solution is a Krebs-Cs⁺ solution. The dashed line indicates zero current. Data were obtained from ramp protocols by averaging the current in a small voltage window around +100 and −100 mV. (F) Current-voltage relations from voltage ramps at the times indicated in (E).

Figure 5

(A) I_GCC traces in control conditions in response to 25 ms voltage steps to potentials from −105 to +195 mV (spaced 25 mV), applied from a holding potential of 0 mV. Sampling interval was 0.1 ms. The arrow indicates zero current. (B) Currents recorded from the same cell as in (A) in the presence of 1.2 μM chlorotoxin, using the same protocol as in (A). (C) Current traces in control conditions (100% extracellular chloride), using the same protocol as in (A). (D) Currents recorded from the same cell as in (C) in the presence of 10% extracellular chloride, using the same protocol as in (A).

Figure 6

(A) I_GCC traces in control conditions in response to 25 ms voltage steps to potentials from −105 to +195 mV (spaced 25 mV), applied from a holding potential of 0 mV. Sampling interval was 0.1 ms. The arrow indicates zero current. (B) Currents recorded from the same cell as in (A) in the presence of 10 mM TEA, using the same protocol as in (A). (C) Currents after washing out the TEA effect, using the same protocol as in (A). (D) Current traces representing the difference current between control current and the current blocked by 10 mM TEA, obtained from the data shown in (A) and (B). (E) Current traces using the same protocol and bath solution as in (A), but with a pipette containing TEACl instead of KCl. (F) Currents recorded from the same cell as in (E) in the presence of 10 mM TEA, using the same protocol as in (A).

Figure 7

(A) Tail currents through GCC in control conditions (100% extracellular chloride), in response to the following protocol: the cell was held at 0 mV, pulsed every 2 s to 180 mV for 25 ms (not shown, sampling interval 0.05 ms) and then stepped (for 12.5 ms, sampling interval 0.02 ms) to various voltages ranging from +120 to −120 mV in −20 mV increments. (B) Tail currents from the same cell in 10% extracellular chloride, using the same protocol as described in (A). (C) Voltage dependence of the amplitude of the tail currents from (A) and (B), assessed from the peak currents measured between 1 and 1.5 ms from the beginning of the step, and corrected for leak current by subtracting the mean current at the end of the voltage step. (D) Tail currents through GCC from another cell in control conditions (5 mM KCl) in response to the voltage protocol described in (A). (E) GCC tail currents from the same cell in high K⁺ conditions (130 mM). (F) I-V curve of the tail currents measured in control conditions (D) and in high K⁺ (E), using the same procedure as in (C).