PHOSPHORYLATION-REGULATED LOW-CONDUCTANCE CL-CHANNELS IN A HUMAN PANCREATIC DUCT CELL-LINE

A low-conductance Cl- channel has been identified in the apical membra ne of the human pancreatic duct cell Capan-1 using patch-clamp techniq ues. Cell-attached channels were activated by the vasoactive intestina l polypeptide (VIP, 0.1 mu mol/l), dibutyryl-adenosine 3',5'-cyclic mo nophosphate (db-cAMP, 1 mmol/l), 8-bromo adenosine 3',5'-cyclic monoph osphate (8-Br-cAMP, 1 mmol/l), 3-isobutyl-1-methyl-xanthine (IBMX, 100 mu mol/l) and forskolin (10 mu mol/l). No channel activity was observ ed in non-stimulated control cells. In both cell-attached and excised inside-out patches, the channel had a linear current/voltage relations hip 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 subs titution led to the selectivity sequence CI- > I- much greater than > HCO3- > gluconate. In inside-out excised patches, the channel activity declined spontaneously within a few minutes. Reactivation of silent e xcised channels was achieved by adding protein kinase A (PKA, in the p resence of ATP, cAMP and Mg2+). Conversely, active channels were silen ced in the presence of alkaline phosphatase. The PKA-activated Cl- cha nnel was 4,4'-diisothiocyanatostilbene-2,2'-disulphonic acid (DIDS, 10 0 mu mol/l) and 4-acetamido-4'isothi ocyanatostilbene-2,2'-disulphonic acid (SITS, 100 mu mol/l) insensitive, but was blocked by diphenylami ne-2-carboxylic acid (DPC, 100 mu mol/l). These results demonstrate th at the apical low-conductance Cl- channel in Capan-1 is regulated on-c ell by VIP receptors via cAMP and off-cell by PKA and phosphatases. Th ey provide evidence that this channel is closely related to the cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channel.