Figure 3.

LPA-induced membrane depolarization and inward currents. (A) LPA-stimulated N1E-115 cells undergo transient Cl⁻-mediated membrane depolarization as measured by a voltage-sensitive dye (see Materials and Methods). Indicated RNA interference or drug treatments correspond to those shown in Figure 2A (n > 100) (transfection marker CFP-H2B). End-of-experiment calibration was done by using 150 mM KCl, which eliminates the transmembrane potential. (B) Latrunculin A (1 μM; up to 1 h pretreatment) does not affect LPA-induced membrane depolarization, whereas it abolishes CLIC4 translocation (see Figure 2B). Recording is representative of eight experiments. (C) Representative patch-clamp recordings of inward Cl⁻ currents in LPA-stimulated N1E-115 cells. Membrane potential was clamped at −60 mV (for details, see Postma et al., 1996a). Black trace, untreated cells. Red trace, CLIC4-depleted cells (three RNA interference targeting constructs). The bar diagram shows peak amplitudes (mean ± SEM). Transmembrane currents were corrected for membrane conductance, which is a measure for total cell surface area (pA/pF). Bottom, Western blots showing CLIC4 levels in control and knockdown N1E-115 cells. Actin was used as loading control.