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), NO3- (1.8), ClO4- (1.7), Br- (1.7), I- (1.6), Cl- (1.0), HCO3- (0.5), gluconate- (0.2). Bath additions of NPPB, flufenamate, glibenclamide (all 100 microM) and DIDS (500 microM) produced varying degrees of block of OR currents with NPPB being the most potent (IC50 of approximately 50 microM) 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), NO3- (1.9), Br- (1.4), I- (1.1), Cl- (1.0), ClO4- (0.5), HCO3- (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 microM 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.

Extracellular zinc stimulates a calcium-activated chloride conductance through mobilisation of intracellular calcium in renal inner medullary collecting duct cells

“…1e). This resulted in a mean shift in E rev of 16.4±2 mV, giving a P Cation /P Cl of 0.25 (n=3), similar to that which has previously been reported for I CLCA [28,31], indicating that the zinc-stimulated whole-cell current was predominantly Cl − selective. Next, the sensitivity of I CLCA to extracellular zinc was investigated.…”

“…We now show that I CLCA is stimulated by extracellular zinc. Under basal conditions, 74% of cells (64/87) displayed whole-cell currents, which were outwardly rectifying and time independent at voltages between −100 and +80 mV similar to I CLCA previously reported in this cell line [28,31]. Addition of ZnCl 2 (400 μM) to the bath solution resulted in an increase in whole-cell current density at ±60 mV, reaching a plateau after 5 min, which was partially reversible after the removal of ZnCl 2 from the bathing solution (Fig.…”

“…mIMCD-3 cells have a large basally active Ca 2+ -sensitive Cl − conductance (I CLCA ) which is stimulated by extracellular ATP, by kinins and by increases in extracellular Ca 2+ through a CaSR-independent mechanism [28,31]. In the present study, we sought to investigate whether mIMCD-3 cells contain an atypical divalent cation receptor by studying the effect of extracellular Zn 2+ on I CLCA and intracellular Ca 2+ .…”

General – Membrane Transport

“…We are using a mouse inner medullary cell line (mIMCD-3) as an appropriate model to study the Clconductances in this nephron segment. Our previous studies, using the fast whole-cell configuration of the patch-clamping technique, showed that 78 % of cells studied had substantial resting basal Cl-conductances (Shindo et al 1996), similar to those seen in other secretory cells after secretagogue stimulation. The purpose of this report was to determine if the high basal conductances in mIMCD3 cells were due to the loss of intracellular factors that occurs with this method.…”

Urinary Stone Formation: Dent’s Disease Moves Understanding Forward

“…Thus activation of this conductance will directly promote entry/exit of anions from the tubule lumen. It is likely that there is a finite permeability of this conductance even to large organic anions such as gluconate [7]. The CLCA protein is a novel candidate for modulation of the milieu of the collecting duct, and its dysregulation may lead to stone formation.…”