Kinetics and regulation of a Ca2+-activated Cl- conductance in mouse renal inner medullary collecting duct cells

• Published in: American journal of physiology. Renal physiology 2004-04, Vol.286 (4), p.F682-F692
• Main Authors: Boese, S H, Aziz, O, Simmons, N L, Gray, M A
• Format: Article
• Language: English
• Subjects: Animals; Bestrophins; Calcium - pharmacokinetics; Cells, Cultured; Chloride Channels - metabolism; Chlorides - metabolism; Cytosol - metabolism; Eye Proteins - metabolism; Ion Channel Gating - physiology; Ion Channels; Kidney Medulla - cytology; Kidney Medulla - metabolism; Kidney Tubules, Collecting - cytology; Kidney Tubules, Collecting - metabolism; Kinetics; Mice; Patch-Clamp Techniques
• ISSN: 1931-857X
• DOI: 10.1152/ajprenal.00123.2003

Using the whole cell patch-clamp technique, a Ca2+-activated Cl- conductance (CaCC) was transiently activated by extracellular ATP (100 microM) in primary cultures of mouse inner medullary collecting duct (IMCD) cells and in the mouse IMCD-K2 cell line. ATP also transiently increased intracellular Ca2+ concentration ([Ca2+]i) from 100 nM to peak values of approximately 750 nM in mIMCD-K2 cells, with a time course similar to the ATP-induced activation and decay of the CaCC. Removal of extracellular Ca2+ had no major effect on the peak Cl- conductance or the increase in [Ca2+]i induced by ATP, suggesting that Ca2+ released from intracellular stores directly activates the CaCC. In mIMCD-K2 cells, a rectifying time- and voltage-dependent current was observed when [Ca2+]i was fixed via the patch pipette to between 100 and 500 nM. Maximal activation occurred at approximately 1 microM [Ca2+]i, with currents losing any kinetics and displaying a linear current-voltage relationship. From Ca2+-dose-response curves, an EC50 value of approximately 650 nM at -80 mV was obtained, suggesting that under physiological conditions the CaCC would be near fully activated by mucosal nucleotides. Noise analysis of whole cell currents in mIMCD-K2 cells suggests a single-channel conductance of 6-8 pS and a density of approximately 5,000 channels/cell. In conclusion, the CaCC in mouse IMCD cells is a low-conductance, nucleotide-sensitive Cl- channel, whose activity is tightly coupled to changes in [Ca2+]i over the normal physiological range.