Ca2؉ -activated Cl ؊ channels play important diverse roles from fast block to polyspermy to olfactory transduction, but their molecular identity has not been firmly established. By searching sequence databases with the M2 pore domain of ligand-gated anion channels, we identified potential Ca 2؉ -activated Cl ؊ channels, which included members of the bestrophin family. We cloned two bestrophins from Xenopus oocytes, which express high levels of Ca 2؉ -activated Cl ؊ channels. The Xenopus bestrophins were expressed in a variety of tissues. We predict that bestrophin has six transmembrane domains with the conserved RFP domain playing an integral part in ionic selectivity. When Xenopus bestrophins were heterologously expressed in human embryonic kidney-293 cells, large Ca 2؉ -activated Cl ؊ currents were observed. The currents are voltage-and time-independent, do not rectify, have a K d for Ca 2؉ of ϳ210 nM, and exhibit a permeability ratio of I ؊ > Br ؊ > Cl ؊ > > aspartate. The W93C and G299E mutations produce non-functional channels that exert a dominant negative effect on wild type channels. We conclude that bestrophins are the first molecularly identified Cl ؊ channels that are dependent on intracellular Ca 2؉ in a physiological range.

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).