Characterization of constitutive and acid-induced outwardly rectifying chloride currents in immortalized mouse distal tubular cells

Thiazides block Na⁺ reabsorption while enhancing Ca²⁺ reabsorption in the kidney. As previously demonstrated in immortalized mouse distal convoluted tubule (MDCT) cells, chlorothiazide application induced a robust plasma membrane hyperpolarization, which increased Ca²⁺ uptake. This essential thiazide-induced hyperpolarization was prevented by the Cl⁻ channel inhibitor 5-Nitro-2-(3-phenylpropylamino) benzoic acid (NPPB), implicating NPPB-sensitive Cl⁻ channels, however the nature of these Cl⁻ channels has been rarely described in the literature. Here we show that MDCT cells express a dominant, outwardly rectifying Cl⁻ current at extracellular pH 7.4. This constitutive Cl⁻ current was more permeable to larger anions (Eisenman sequence I; I⁻ > Br⁻ ≥ Cl⁻) and was substantially inhibited by > 100 mM [Ca²⁺]_o, which distinguished it from ClC-K2/barttin. Moreover, the constitutive Cl⁻ current was blocked by NPPB, along with other Cl⁻ channel inhibitors (4,4′-diisothiocyanatostilbene-2,2′-disulfonate, DIDS; flufenamic acid, FFA). Subjecting the MDCT cells to an acidic extracellular solution (pH < 5.5) induced a substantially larger outwardly rectifying NPPB-sensitive Cl⁻ current. This acid-induced Cl⁻ current was also anion permeable (I⁻ > Br⁻ > Cl⁻), but was distinguished from the constitutive Cl⁻ current by its rectification characteristics, ion sensitivities, and response to FFA. In addition, we have identified similar outwardly rectifying and acid-sensitive currents in immortalized cells from the inner medullary collecting duct (mIMCD-3 cells). Expression of an acid-induced Cl⁻ current would be particularly relevant in the acidic IMCD (pH < 5.5). To our knowledge, the properties of these Cl⁻ currents are unique and provide the mechanisms to account for the Cl⁻ efflux previously speculated to be present in MDCT cells.