Characterization of Ca2+-activated Cl- currents in mouse kidney inner medullary collecting duct cells.
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 microM) 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 approximately 0.4 microM at +100 mV and approximately 1.0 microM 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 microM. 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).
Qu
Zhiqiang
Z
Department of Cell Biology, Emory Univ. School of Medicine, 615 Michael St., Atlanta, GA 30322-3030, USA. zqu@cellbio.emory.edu
Wei
Raymond W
RW
Hartzell
H Criss
HC
D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid / Q000494:pharmacology; D000818:Animals; D000873:Anthracenes / Q000494:pharmacology; D055592:Biophysical Phenomena; D001703:Biophysics; D002118:Calcium / Q000378:metabolism*; D002121:Calcium Channel Blockers / Q000494:pharmacology; D002273:Carcinogens / Q000494:pharmacology; D002460:Cell Line; D018118:Chloride Channels / Q000378:metabolism*; D002712:Chlorides / Q000378:metabolism; D003600:Cytosol / Q000378:metabolism; D004229:Dithiothreitol / Q000494:pharmacology; D007679:Kidney Medulla / Q000166:cytology / Q000378:metabolism; D007685:Kidney Tubules, Collecting / Q000166:cytology / Q000378:metabolism*; D008564:Membrane Potentials / Q000187:drug effects / Q000502:physiology; D051379:Mice; D009865:Oocytes / Q000166:cytology / Q000378:metabolism; D018408:Patch-Clamp Techniques; D013629:Tamoxifen / Q000494:pharmacology; D014982:Xenopus laevis; D062367:ortho-Aminobenzoates / Q000494:pharmacology
D000873:Anthracenes; D002121:Calcium Channel Blockers; D002273:Carcinogens; D018118:Chloride Channels; D002712:Chlorides; D062367:ortho-Aminobenzoates; D013629:Tamoxifen; C010030:9-anthroic acid; C001209:fenamic acid; D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; D002118:Calcium; D004229:Dithiothreitol