Characterization of Whole Cell Chloride Conductances in a Mouse Inner Medullary Collecting Duct Cell Line mIMCD-3

Abstract.

The chloride conductance of inner medullary collecting duct cells (mIMCD-3 cell line) has been investigated using the whole cell configuration of the patch clamp technique. Seventy-seven percent of cells were chloride selective when measured with a NaCl-rich bathing solution and a TEACl-rich pipette solution. Seventy-five percent of chloride-selective cells (90/144) had whole cell currents which exhibited an outwardly-rectifying (OR) current-voltage (I/V) relationship, while the remaining cells exhibited a linear (L) I/V relationship. The properties of the OR and L chloride currents were distinct. OR currents (mean current densities at ±60 mV of 66 ± 5 pA/pF and 44 ± 3 pA/pF), were time- and voltage-independent with an anion selectivity (from calculated permeability ratios) of SCN⁻ (2.3), NO⁻ ₃ (1.8), ClO⁻ ₄ (1.7), Br⁻ (1.7), I⁻ (1.6), Cl⁻ (1.0), HCO⁻ ₃ (0.5), gluconate⁻ (0.2). Bath additions of NPPB, flufenamate, glibenclamide (all 100 μm) and DIDS (500 μm) produced varying degrees of block of OR currents with NPPB being the most potent (IC₅₀ of approximately 50 μm) while DIDS was the least effective. Linear chloride currents had similar current densities to the OR chloride currents and were also time- and voltage-independent. The anion selectivity sequence was SCN⁻ (2.5), NO⁻ ₃ (1.9), Br⁻ (1.4), I⁻ (1.1), Cl⁻ (1.0), ClO⁻ ₄ (0.5), HCO⁻ ₃ (0.5), gluconate⁻ (0.3). In contrast to the OR conductance, glibenclamide was the most potent and DIDS the least potent blocker of L currents. An IC₅₀ of >100 μm was observed for NPPB block. Neither OR of L chloride currents were affected by acutely or chronically increased intracellular cAMP and were not affected when intracellular Ca²⁺ levels were increased or decreased. The molecular identity and physiological role of OR and linear currents in mIMCD-3 cells are discussed.