FIGURE 4.

Role of ATP release and P2X4 activation on VSOR Cl⁻ currents. A, bulk ATP released from HTC cells measured after 5 min of exposure to 33% hypotonicity (empty bars) or 200 μm H₂O₂ (light gray bars). n = 3 independent experiments. *, p < 0.05 respect to basal release. B, representative current record of a cell kept at a holding potential of −80 mV exposed to 10 μm ATP. The same cell exposed to a second puff of ATP in the presence of 100 μm suramin. After suramin wash-out, partial recovery of the current is observed. ATP was delivered for the time indicated by the bar using a puffing pipette located nearby the cell. For these experiments, a 15% hypertonic solution was uses to avoid any contribution from volume-sensitive Cl⁻ currents. C, representative current record of a cell kept at a holding potential of −80 mV exposed to 10 μm ATP in a cell overexpressing a dominant-negative mutant for P2X4 (P2X4 K313A). The same cell exposed to a second puff of ATP after 15 min of preincubation with 4 μm ivermectin. n = 3–4 independent experiments. D, representative experiments showing the time course VSOR Cl⁻ currents evoked by 33% hypotonicity in cells overexpressing P2X4 K313A in the absence (●) or in the presence (○) of 2 mm external Ca²⁺. Currents were measured at 80 mV every 7 s and normalized by cell capacitance. E, summary of the data presented in D for normalized maximal currents at 80 mV (left axis) and half-time for current onset (right axis) obtained from n = 6 independent experiments. *, p < 0.05 compared with control conditions.