Characterization of a Family of Scorpion Toxins Modulating Ca2+-Activated Cl- Current in Vascular Myocytes

Abstract

The pharmacology of calcium-activated chloride current is not well developed. Peptides from scorpion venom present potent pharmacological actions on ionic conductance used to characterize the function of channels but can also be helpful to develop organic pharmacological tools. Using electrophysiological recording coupled with calcium measurement, we tested the potent effect of peptides extracted from Leuirus quinquestratus quinquestratus venom on the calcium-activated chloride current expressed in smooth muscle cells freshly dissociated from rat portal veins. We identified one peptide which selectively inhibited the chloride conductance without effects on either calcium signaling or calcium and potassium currents expressed in this cell type. The synthetic peptide had the same affinity, but the chemical modification of the amino acid sequence altered the efficiency to inhibit the calcium-activated chloride conductance.

Keywords: animal toxins; calcium-activated chloride channel; scorpion venom; vascular smooth muscle.

Figure 1

HPLC chromatogram showing (a) Lqh 2-2, (b) Lqh 7-1 and (c) Lqh 8-6 peptides selected to be tested on vascular smooth muscle cells from rat portal veins.

Figure 2

Full sequence of Lqh peptides and comparison with other closed known sequences described in [34]. The color code was applicated on all figures. Lqh toxins and GaTx1, known as CFTR inhibitors, were from Leuirus quinquestratus hebraeus, Lqq-CTX (Chlorotoxin) from Leuirus quinquestratus quinquestratus; AmmP2 from Androctonus mauretanicus mauretanicus; BsTx from Buthus sindicus; BeI from Buthus cupeus; ButaIT from Mesobuthus tamulus; BtITx3 from Hottentotta tamulus; BmKCl1 and Bm12B from Mesobuthus martensii; and ClTx from Odontobuthus doriae.

Figure 3

(a) typical immunolabelling with anti-TMEM16A revealed by confocal microscopy in rat portal vein slice. The experiments were reproduced on dissociations from three different rats. (b) Effect of Lqh 7-1 (1 µM) on typical Ca²⁺-activated Cl⁻ current activated by caffeine application in control conditions (black) and in presence of the peptide (blue). (c) Effect concentration curves of Lqh 2-2 (green), native Lqh 7-1 (dark blue), synthetic Lqh 7-1 (blue), chlorotoxin (red) and Lqh 8-6 (orange). n = 7–31 cells per concentrations reported in the graph (c), cells were provided from five different dissociations/rats.

Figure 4

Effect of Lqh 7-1 peptide (0.1 and 0.5 µM) (a) on a representative Ca²⁺-activated current in presence of Cesium chloride to inhibit K⁺ currents; two different traces with two different concentration of Lqh 7-1 were superimposed (b) on norepinephrine-induced Ca²⁺ responses, (c) on voltage activated Ba²⁺ current through voltage gated Ca²⁺ channels as described by Loirand et al. [35] and (d) on a voltage-activated K⁺ current activated by depolarization. Dark blue traces illustrated measurements were recorded in presence of peptide Lqh 7-1 (0.5 µM).

Figure 5

(a) Model structure of the native Lqh7-1 obtained by AlphaFold [40], (b) sequences and inhibition of Ca^2+-activated chloride current evoked by caffeine application on vascular smooth muscle cells. (c) Representative Ca^2+-activated currents evoked by caffeine application in control conditions (black) and after 10 min of the application in the perfusion bath of the Lqh-7.1 toxin mutants (in blue, n = 9–15 cells were recorded in the absence and presence of 1 µM of toxin mutants). Traces were superimposed and the pipette solution contains cesium chloride to inhibit K+ currents. The lightning bolts indicate the point where the mutations are made. The PDB files of the models are given in the Supplementary Materials File S1.