Short-circuit current measurement.

T84 cells were mounted in Ussing chambers and bathed in symmetrical HCO₃^–-buffered solution containing (in mM) 120 NaCl, 5 KCl, 1 MgCl₂, 1 CaCl₂, 10 d-glucose, 5 HEPES, and 25 NaHCO₃ (pH 7.4). The solutions were aerated with 95% O₂/5% CO₂ and maintained at 37°C. For measurement of basolateral K⁺ conductance, a mucosal-to-serosal K⁺ gradient was established using solutions containing K⁺ as the major charge-carrying ion. The apical solution contained (in mM) 142.5 K-gluconate, 1.25 CaCl₂, 0.40 MgSO₄, 0.43 KH₂PO₄, 0.35 Na₂HPO₄, 10 HEPES, and 5.6 d-glucose, pH 7.4. In basolateral solution 142.5 mM K-gluconate was replaced by 5.4 mM K-gluconate and 136.9 mM N-methylglucamine, and the apical membrane was permeabilized with 20 μM amphotericin B (32). For measurement of apical Cl^– conductance, a basolateral-to-apical Cl^– gradient was applied. The basolateral solution contained (in mM) 120 NaCl, 1 MgCl₂, 1 CaCl₂, 10 d-glucose, 5 HEPES, and 25 NaHCO₃ (pH 7.4). In the apical solution 120 mM NaCl was replaced by 5 mM NaCl and 115 mM Na-gluconate, and the basolateral membrane was permeabilized with 250 μg/ml amphotericin B (62). Short-circuit current was measured using an EVC4000 multichannel voltage clamp (World Precision Instruments). For intestinal short-circuit current measurement, CD1 mice were anesthetized with isoflurane. The ileum was removed, washed with ice-cold Krebs buffer, and opened along the mesenteric border, and a full-thickness layer was mounted in a micro-Ussing chamber (area 0.7 cm², World Precision Instruments). Hemichambers were filled with oxygenated Krebs-Ringer bicarbonate solution.