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1 Institute for Biochemistry & Biology, University Potsdam, Potsdam, Germany; School of Cell and Molecular Biosciences, University Medical School, Newcastle upon Tyne, United Kingdom
2 Laboratory of Signal Transduction, NIEHS, Durham, North Carolina, USA; School of Cell and Molecular Biosciences, University Medical School, Newcastle upon Tyne, United Kingdom
3 School of Cell and Molecular Biosciences, University Medical School, Newcastle upon Tyne, United Kingdom
* To whom correspondence should be addressed. E-mail: m.a.gray{at}ncl.ac.uk.
Using the whole cell patch clamp technique a Ca2+-activated chloride conductance (CaCC) was transiently activated by extracellular ATP (100 µM) in primary cultures of mouse 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 similar time course 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 100-500 nM. Maximal activation occurred at ~1 µM [Ca2+]i with currents losing any kinetics and displaying a linear I/V relationship. From Ca2+-dose response curves an EC50 value of ~650 nM at -80mV 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 suggest a single channel conductance of 6-8 pS, and a density of ~5000 channels per 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.
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