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

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 pH7.4. This constitutive Cl^- current was more permeable to larger anions (Eisenman sequence I; I^->Br^-≥Cl^-) and was substantially inhibited by >100mM [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.