2.4. Ussing chambers experiments

Confluent kAE1-HA expressing mIMCD3 cells were grown for 10 days on 0.45 μm semi-permeable Transwell filters (Costar, Cat # 07200225). One day before the start of the experiment, these cells were transiently transfected using lipofectamine 3000 with cDNA encoding either WNK4-WT-HA or WNK4-D321A-HA. Expression of kAE1 protein was induced by the addition of 0.5 μg/ml of doxycycline to the cells or cells were kept untreated. The filters were then mounted in Ussing chambers maintained at 37 °C in solution A (145 mM NaCl, 1 mM CaCl₂, 1 mM MgCl₂, 10 mM glucose, and 10 mM HEPES, pH 7.4), followed by current clamping with a DVC 1000 I/V clamp (World Precision Instruments, Sarasota, FL) and filling electrodes with 3 M KCl. PowerLab (ADInstruments, Colorado Springs, CO) and Chart 4.0 software were used to record the data. We started by applying 90 μA pulses across the epithelial monolayer, followed by induction of a dilution potential by switching basolateral solution A to an iso-osmotic solution B with reduced NaCl to 80 mM (Solution B: 80 mM NaCl, 130 mM mannitol, 1 mM CaCl₂, 1 mM MgCl₂, 10 mM glucose, and 10 mM HEPES, pH 7.4). Transepithelial electrical resistance (TEER), ion permeability ratios and absolute permeabilities of the epithelium to sodium and chloride were then calculated with the Goldman-Hodgkin Katz and Kimizuka Koketsu equations, as previously published [3,22,23].