Fig 2.
SNAP-23 regulates CFTR-mediated chloride currents in HT29-Cl.19A epithelial cells. Whole-cell patch-clamp experiments and capacitance measurements were performed as described in Materials and Methods. (A) Inhibition of cAMP-dependent currents by GST–SNAP-23 (40 μg/ml or 0.8 μM) but not by GST–vesicle-associated membrane protein-2 (VAMP-2) (40 μg/ml or 1.1 μM). (B) Anti-SNAP-23 IgG or Fab fragment (20 μg/ml) increased cAMP-activated currents whereas preimmune IgG (20 μg/ml) had no effect. Note change in scale compared with A. Currents were recorded at +110 mV. (Inset) Current–voltage relationship for the Fab-potentiated currents in the presence and absence of 1 mM DPC. Data are expressed as mean ± SEM. Asterisk (*) indicates P < 0.05 relative to untreated control. (C) Whole-cell currents in the presence of anti-SNAP-23 IgG (20 μg/ml) showing time independence, DIDS insensitivity, and DPC sensitivity characteristic of CFTR-mediated currents. (D) Representative simultaneous recordings of cell capacitance (Cm) and membrane conductance (Gm). Each pair of traces derives from an individual HT29-Cl.19A cell. The anti-pan dynamin IgG (30) antibody and anti-SNAP-23 IgG were added at 100 and 20 μg/ml, respectively. (E) Mean changes in Cm. (F) Mean changes in Gm.