Kinetics and regulation of a Ca2+-activated Cl- conductance in mouse renal inner medullary collecting duct cells.
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.
Boese
S H
SH
School of Cell and Molecular Bioscience, Univ. Medical School, Newcastle Upon Tyne, NE2 4HH, UK.
Aziz
O
O
Simmons
N L
NL
Gray
M A
MA
D000818:Animals; D000075002:Bestrophins; D002118:Calcium / Q000493:pharmacokinetics*; D002478:Cells, Cultured; D018118:Chloride Channels / Q000378:metabolism*; D002712:Chlorides / Q000378:metabolism*; D003600:Cytosol / Q000378:metabolism; D005136:Eye Proteins / Q000378:metabolism; D015640:Ion Channel Gating / Q000502:physiology; D007473:Ion Channels; D007679:Kidney Medulla / Q000166:cytology / Q000378:metabolism*; D007685:Kidney Tubules, Collecting / Q000166:cytology / Q000378:metabolism*; D007700:Kinetics; D051379:Mice; D018408:Patch-Clamp Techniques
C491856:Best1 protein, mouse; D000075002:Bestrophins; D018118:Chloride Channels; D002712:Chlorides; D005136:Eye Proteins; D007473:Ion Channels; D002118:Calcium