Phosphorylation-regulated low-conductance Cl- channels in a human pancreatic duct cell line.
A low-conductance Cl- channel has been identified in the apical membrane of the human pancreatic duct cell Capan-1 using patch-clamp techniques. Cell-attached channels were activated by the vasoactive intestinal polypeptide (VIP, 0.1 mumol/l), dibutyryl-adenosine 3',5'-cyclic monophosphate (db-cAMP, 1 mmol/l), 8-bromo adenosine 3',5'-cyclic monophosphate (8-Br-cAMP, 1 mmol/l), 3-isobutyl-1-methyl-xanthine (IBMX, 100 mumol/l) and forskolin (10 mumol/l). No channel activity was observed in non-stimulated control cells. In both cell-attached and excised inside-out patches, the channel had a linear current/voltage relationship and a unitary conductance of 9 pS at 23 degrees C and 12 pS at 37 degrees C. Its opening probability was not voltage dependent although pronounced flickering was induced at negative potentials. Anionic substitution led to the selectivity sequence Cl- > I- >> > HCO3- > gluconate. In inside-out excised patches, the channel activity declined spontaneously within a few minutes. Reactivation of silent excised channels was achieved by adding protein kinase A (PKA, in the presence of ATP, cAMP and Mg2+). Conversely, active channels were silenced in the presence of alkaline phosphatase. The PKA-activated Cl- channel was 4,4'-diisothiocyanatostilbene-2,2'-disulphonic acid (DIDS, 100 mumol/l) and 4-acetamido-4'-isothiocyanatostilbene-2,2'-disulphonic acid (SITS, 100 mumol/l) insensitive, but was blocked by diphenylamine-2-carboxylic acid (DPC, 100 mumol/l). These results demonstrate that the apical low-conductance Cl- channel in Capan-1 is regulated on-cell by VIP receptors via cAMP and off-cell by PKA and phosphatases.(ABSTRACT TRUNCATED AT 250 WORDS)
Becq
F
F
Laboratoire de Neurobiologie Cellulaire, CNRS, Marseille, France.
Hollande
E
E
Gola
M
M
D015056:1-Methyl-3-isobutylxanthine / Q000494:pharmacology; D015124:8-Bromo Cyclic Adenosine Monophosphate / Q000494:pharmacology; D000469:Alkaline Phosphatase / Q000494:pharmacology; D000838:Anions; D001692:Biological Transport / Q000187:drug effects; D003994:Bucladesine / Q000494:pharmacology; D002460:Cell Line; D002462:Cell Membrane / Q000502:physiology; D018118:Chloride Channels / Q000187:drug effects / Q000502:physiology*; D005576:Colforsin / Q000494:pharmacology; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism / Q000494:pharmacology; D004553:Electric Conductivity; D006801:Humans; D010183:Pancreatic Ducts / Q000502:physiology*; D010766:Phosphorylation; D014660:Vasoactive Intestinal Peptide / Q000494:pharmacology
D000838:Anions; D018118:Chloride Channels; D005576:Colforsin; D015124:8-Bromo Cyclic Adenosine Monophosphate; D014660:Vasoactive Intestinal Peptide; D003994:Bucladesine; D017868:Cyclic AMP-Dependent Protein Kinases; D000469:Alkaline Phosphatase; D015056:1-Methyl-3-isobutylxanthine