α-Toxin–permeabilized HBE monolayers exhibit constitutive and forskolin-stimulated chloride currents and voltage-dependent blocker effects. (A) Short-circuit current measurements performed on three separate filters demonstrate GlyH-101 and glibenclamide inhibition properties. With a mucosal to serosal chloride gradient of 48 mM (Eq_Cl = −13 mV), constitutive chloride currents from mucosa to serosa were evident after permeabilization, and forskolin stimulation tripled the current. GlyH-101 rapidly inhibited the stimulated current (blue trace), whereas glibenclamide inhibition was slower and less effective (black trace). GlyH-101 was equally effective at inhibiting the constitutive current before forskolin stimulation (red trace). The forskolin-stimulated current after GlyH-101 block was small, but forskolin still significantly shifted the R_IV, consistent with activation of CFTR (see Results). Bars below the traces indicate additions to the basolateral chamber, whereas those above indicate additions to the apical chamber. Permeabilization with α-toxin required ∼15 min; ouabain was added to inhibit Na-K-ATPase, whereas ATP was added to replenish intracellular stores (Supplemental text). Solutions were bicarbonate free, and recordings were started within 2 min of filter submersion. Representative tracings of at least four filters per blocker are shown. (B) Same tracings as in A but including the I-V measurements, consisting of bipolar pulses from ±10 to ±60 mV in 10-mV steps. Pulses were simultaneously applied to two filters for the traces shown. I-V measurements were performed after permeabilization (basal), forskolin stimulation (fsk), and blocker inhibition (fsk+blocker). Although the I-V responses overlapped for the basal and fsk measurements, differences in the voltage dependence of the two blockers were evident in the third measurement. Cation channel blockers (A) were added to minimize contributions from ENaC and potassium channels to the I-V response. The permeabilization phase of the recording was removed for clarity. (C) The I-V curves measured for the forskolin-stimulated monolayer treated with glibenclamide (black) demonstrate the shift from linear (open symbols) to OR on blocker addition (solid triangles), whereas the I-V curves measured for the stimulated monolayer treated with GlyH-101 (blue) demonstrate the shift from linear (open symbols) to IR on blocker addition (blue triangles).