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

School of Cell and Molecular Biosciences, University Medical School, Newcastle Upon Tyne, NE2 4HH, United Kingdom

Submitted 27 March 2003 ; accepted in final form 12 December 2003

Using the whole cell patch-clamp technique, a Ca²⁺-activated Cl^- conductance (CaCC) was transiently activated by extracellular ATP (100 µM) 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 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 1 µM [Ca²⁺]_i, with currents losing any kinetics and displaying a linear current-voltage relationship. From Ca²⁺-dose-response curves, an EC₅₀ value of 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 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.

renal collecting duct; calcium-activated chloride conductance; intracellular calcium

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