ATP-sensitive K+ channels regulated by intracellular Ca2+ and phosphorylation in normal (T84) and cystic fibrosis (CFPAC-1) epithelial cells.
The elementary K+ conductance activated by the cAMP or the Ca2+ second messenger pathways was investigated in the model salt-secreting epithelium, the human T84 cell line. Under Cl(-)-free conditions, an inwardly rectifying whole-cell K+ current was evoked by either forskolin 10 (mumol/l) or acetylcholine 1 (mumol/l) and blocked by extracellular charybdotoxin 10 (nmol/l). In the cell-attached mode, both secretory agonists induced the opening of a channel showing inward rectification with a unitary chord conductance of 36.8 +/- 2.5 pS (n = 26) for inward currents. In inside-out patches, a 35-pS inwardly rectifying K+ channel that corresponded to the channel recorded in the cell-attached configuration was recorded in the presence of 0.3 mumol/l free Ca2+ at the inner side of the membrane. This channel was blocked by Ba2+ (5 mumol/l) and by charybdotoxin (50 nmol/l). Its open probability was enhanced by intracellular Ca2+ with and EC50 of 0.25 mumol/l and strongly reduced by intracellular MgATP with an IC50 of 600 mumol/l. In the continuous presence of ATP, the channel activity was consistently increased by 125 kU/l catalytic subunit of cAMP-dependent protein kinase. In the cystic fibrosis pancreatic duct cell line CFPAC-1, a K+ channel was also recorded, with similar characteristics and regulation as the 35-pS channel in T84 cells. We conclude that an ATP-sensitive K+ channel regulated by intracellular Ca2+ and phosphorylation supports the main K+ current activated by secretory agonists in normal cystic fibrosis cell lines.(ABSTRACT TRUNCATED AT 250 WORDS)
Roch
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Laboratoire de Physiologie Cellulaire, URA CNRS 1121, Université Paris XI, Orsay, France.
Baró
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Hongre
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Escande
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D000109:Acetylcholine / Q000494:pharmacology; D000255:Adenosine Triphosphate / Q000494:pharmacology*; D001464:Barium / Q000494:pharmacology; D001711:Biotransformation / Q000187:drug effects; D002118:Calcium / Q000502:physiology*; D002460:Cell Line; D018999:Charybdotoxin; D005576:Colforsin / Q000494:pharmacology; D003550:Cystic Fibrosis / Q000378:metabolism*; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D004847:Epithelial Cells; D004848:Epithelium / Q000187:drug effects / Q000378:metabolism; D006801:Humans; D015759:Ionomycin / Q000494:pharmacology; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism; D018408:Patch-Clamp Techniques; D010766:Phosphorylation; D015221:Potassium Channels / Q000187:drug effects / Q000378:metabolism* / Q000502:physiology; D012604:Scorpion Venoms / Q000494:pharmacology
C505032:CFTR protein, human; D008565:Membrane Proteins; D015221:Potassium Channels; D012604:Scorpion Venoms; D018999:Charybdotoxin; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D005576:Colforsin; D001464:Barium; D015759:Ionomycin; D000255:Adenosine Triphosphate; D000109:Acetylcholine; D002118:Calcium