Antivenom Evaluation by Electrophysiological Analysis

Abstract

Scorpion stings on humans are medically relevant because they may contain toxins that specifically target ion channels. During antivenom production, pharmaceutical companies must use a large number of experimental animals to ensure the antivenom's efficacy according to pharmacopeia methods. Here we present an electrophysiological alternative for the evaluation of horse antivenoms produced against two species of Moroccan scorpions: Buthus mardochei and Androctonus mauretanicus. Human sodium and potassium channels and acetylcholine nicotinic receptors were analyzed by standard patch-clamp techniques. The results showed that the antivenom is capable of reversing ion current disruption caused by the venom application. We propose the use of this in vitro technique for antivenom evaluation as an alternative to using a large number of live animals.

Keywords: antivenom; electrophysiology; horse immunoglobulin; human ion channels scorpion venoms; patch-clamp; sodium channel.

Figure 1

Characterization of venom effects on sodium channels. Sodium currents produced by hNav 1.3 sodium channels were elicited by using the stimulation protocol as described in (A). This stimulus was applied every 6 s, and the current values were graphed as a function of time, as shown in (C). In control conditions, no channels opened at the sub-threshold potential, whereas all channels opened at the full-activation potential ((B), black trace). Application of 3 µg/mL Buthus mardochei (Bm) venom (Bmv) induced current at the sub-threshold potential (I_shift) and slowed the current inactivation process (I_inactivation). After one-minute venom application, the total current measured at the full-activation potential slightly increased (I_peak), but after more prolonged application, the I_peak started to decrease (respectively, light gray and dark gray lines in (B)). The time-dependence of the venom effect can be seen in panel (C), where the black bar corresponds to venom application.

Figure 2

NA scorpion antivenom protection against Androctonus mauretanicus (Am) venom (Amv) in different sodium channel sub-types. Current values of I_shift, I_peak, and I_inactivation recorded in control are reported and compared with the values of currents recorded after application of Am venom along with different antivenom concentrations. Antivenom at 100 µL/mL completely protects against Am venom effects upon all sodium channels sub-types (panels A–G). In hNav 1.2, hNav 1.5, hNav 1.6, and hNav 1.7, antivenom was also protective at 30 µL/mL (panels B,E–G).

Figure 3

NA scorpion antivenom protection against Bm venom in different sodium channel sub-types. Current values of I_shift, I_peak, and I_inactivation recorded in control are reported and compared with the values of currents recorded after application of Bm venom along with different antivenom concentrations. Antivenom at 100 µL/mL concentration completely protects six sodium channel sub-types against Am venom (panels A–F). For hNav 1.7 channels, 50 µg/mL of venom was used. In this case, the minimum antivenom concentration capable of completely neutralizing this amount of venom was 300 µL/mL (G).

Figure 4

NA scorpion antivenom protection against Am and Bm venom in potassium channels. Potassium channels of the type hERG, hKv 1.1 and hKv1.4 were recorded in control (black traces in A–F) and after application of Bm (A–C) and Am (D–F) venoms (dark gray traces). Both venoms reduce hKv 1.1 current. Antivenom, applied at different concentrations, prevents the blocking effect of both venoms in a dose-dependent manner (light grey traces in C and F).

Figure 5

Am and Bm venoms activity on acetylcholine nicotinic receptor. Currents conducted through nicotinic receptor were elicited by pulse of 10 µM acetylcholine (Ach, black line in A and B). After application of 50 µg/mL of both Am or Bm venoms (grey line in A and B), the current was not significantly modified.