Figure 4.

Characterization of ivermectin effects on recombinant hP2X7 receptors. (A,B) Concentration–response curves of ivermectin (IVM)-induced hP2X7 current potentiation were recorded in low DIC bath solution and at a holding potential of −60 mV by applying repetitive pulses of ATP stimulation (300 µM for 14 s every 2 min for seven times) to allow for run-up of I_ATP and then acutely adding, for 6 s, various concentrations of ivermectin, 4 s after an eighth application of ATP was initiated. Representative responses to the first and seventh ATP application and the eighth ATP/ivermectin co-application are shown superimposed in panel A. (B) Statistical analysis of experiments performed as shown in panel A with superposed mathematical fit according to one or two affinity states of ivermectin. Each data point represents the ratio of the ivermectin-augmented current (I₂) with respect to the current immediately before application of ivermectin (I₁, see panel A), expressed as percentage of I₁ as derived from n= 7–10 independent measurements. (C) Inclusion of 3 µM ivermectin (IVM) in the pipette solution does not prevent or attenuate ivermectin potentiation applied via the bath solution. Shown is the statistical analysis of similar experiments as those in panel A. HEK_hP2X7 cells were alternately microdialysed with either standard pipette solution (n= 8) or with standard pipette solution containing 3 µM ivermectin (n= 7). Note that acute extracellular application of the solvent (DMSO 0.1%) does not substitute for the potentiating effect of ivermectin (n= 10). *P < 0.05, significant differences from the effects of 0.1% DMSO. (D) Current–voltage relationship of ATP-triggered and ivermectin-augmented hP2X7 currents were tested by applying slow voltage ramps. Voltage ramps, extending from −100 to 40 mV, were 1 s in duration and imposed every 2 s on a holding potential of −60 mV. Illustrated are representative leak-subtracted IV traces for the ATP (300 µM)-induced current in the absence (I₁) and presence of ivermectin (3 µM; I₂). Leak subtraction was done by subtracting the ensemble current from 4 V ramps recorded prior to ATP application from that obtained after 4 s in the presence of ATP and that after another 4 s in the additional presence of ivermectin. (E) Ratios of ivermectin-potentiated (I₂) and non-potentiated ATP currents (I₁) are calculated at different command potentials (V_C).