Characterization of Ca2+-activated Cl– currents in mouse kidney inner medullary collecting duct cells

Abstract

Ca2+-activated Cl– (ClCa) channels were characterized biophysically and pharmacologically in a mouse kidney inner medullary collecting duct cell line, IMCD-K2. Whole cell recording was performed with symmetrical N-methyl-d-glucamine chloride (NMDG)-Cl in the intracellular and extracellular solutions, and the intracellular Ca2+ concentration ([Ca2+]i) was adjusted with Ca2+-EGTA buffers. The amplitude of the current was dependent on [Ca2+]i. [Ca2+]i <800 nM strongly activated outwardly rectifying Cl– currents, whereas high Ca2+ (21 μM) elicited time-independent currents that did not rectify. The currents activated at low [Ca2+] exhibited time-dependent activation and deactivation. The affinity of the channel for Ca2+ was voltage dependent. The EC50 for Ca2+ was ∼0.4 μM at +100 mV and ∼1.0 μM at –100 mV. The Cl– channel blocker niflumic acid in the bath equally inhibited both inward and outward currents reversibly, with a Ki = 7.6 μM. DIDS, diphenylamine-2-carboxylic acid, and anthracene-9-carboxylic acid reversibly inhibited outward currents in a voltage-dependent manner. DTT slowly inhibited the currents, but tamoxifen did not. Comparing the biophysical and pharmacological properties, we conclude that IMCD-K2 cells express the same type of ClCa channels as those we have described in detail in Xenopus laevis oocytes (Qu Z and Hartzell HC. J Biol Chem 276: 18423–18429, 2001).