Figure 5. TRPV2 cooperates with BKCa.

(A) BKCa inhibitor IbTx decreases LL-37 induced outward potassium current on MDA-MB-435s cells. Left panel shows examples of currents obtained with I-V protocol on MDA-MB-435s cells in presence (LL-37) or absence (PSS) of the peptide. Currents were measured for each voltage clamp (V) from −90 at +80 mV during 500 ms, every 10 mV increase. Mid panel displays a representative Current-Voltage curves which shown the current density-voltage relationships obtained using ramp protocol recorded in PSS or in presence of LL-37 and with or without pretreatment with 100 nM Ibtx. Right panel represents the compilation of the outward current at the membrane voltage at 0 mV normalized to PSS condition (5 < n < 12). (B) IbTx decreases LL-37 induced Ca2+ influx without additional effect of anti-TRPV2 siRNA (siV2). Fura-2 probe is used to measure Ca²⁺ influx after 2 mM Ca²⁺ application as described in Materials and Methods. The effects of LL-37 and IbTx (100 nM) are evaluated on MDA-MB-435s cells transfected with siRNA against TRPV2 or control siRNA (siCt). Fura-2 fluorescence ratio is normalized against the basal level of each individual experiment. The dotted line shows the normalized basal level and the inhibitory effect of siV2 and/or IbTx is indicated relative to siCtl with LL-37 (n = 13). (C) IbTx decreases LL-37 induced cell migration without additional effect of anti-TRPV2 siRNA (siV2). The inhibitory effect of siV2 and/or IbTx is indicated relative to siCtl with LL-37 (n = 8). (D) BKCa membrane location remains unaltered after application of LL-37. Left panels show the detection of BKCa by immunofluorescence performed on non-permeabilized MDA-MB-435s cells treated or not with LL-37 for 5 min. DAPI is used for nuclear staining. Right panels show the localisation of BKCa on pseudopodia (P) and intracellularly by immunoelectron microscopy. The location of immunogold signals, intracellularly and at pseudopodia (P) is indicated with arrows.