Regulation of an inwardly rectifying K channel in the T84 epithelial cell line by calcium, nucleotides and kinases.
Agonists that elevate calcium in T84 cells stimulate chloride secretion by activating KBIC, an inwardly rectifying K channel in the basolateral membrane. We have studied the regulation of this channel by calcium, nucleotides and phosphorylation using patch clamp and short-circuit current (ISC) techniques. Open probability (Po) was independent of voltage but declined spontaneously with time after excision. Rundown was slower if patches were excised into a bath solution containing ATP (10 microM-5 mM), ATP (0.1 mM)+protein kinase A (PKA; 180 nM), or isobutylmethylxanthine (IBMX; 1 mM). Analysis of event durations suggested that the channel has at least two open and two closed states, and that rundown under control conditions is mainly due to prolongation of the long closed time. Channel activity was restimulated after rundown by exposure to ATP, the poorly hydrolyzable ATP analogue AMP-PNP, or ADP. Activity was further enhanced when PKA was added in the presence of MgATP, but only if free calcium concentration was elevated (400 nM). Nucleotide stimulation and inward rectification were both observed in nominally Mg-free solutions. cAMP modulation of basolateral potassium conductance in situ was confirmed by measuring currents generated by a transepithelial K gradient after permeabilization of the apical membrane using alpha-toxin. Finally, protein kinase C (PKC) inhibited single KBIC channels when it was added directly to excised patches. These results suggest that nonhydrolytic binding of nucleotides and phosphorylation by PKA and PKC modulate the responsiveness of the inwardly rectifying K channel to Ca-mediated secretagogues.
Tabcharani
J A
JA
Department of Physiology, McGill University, Montreal, Quebec, Canada.
Boucher
A
A
Eng
J W
JW
Hanrahan
J W
JW
D015056:1-Methyl-3-isobutylxanthine / Q000494:pharmacology; D000255:Adenosine Triphosphate / Q000032:analysis / Q000494:pharmacology; D001427:Bacterial Toxins / Q000494:pharmacology; D002118:Calcium / Q000032:analysis / Q000378:metabolism*; D002462:Cell Membrane / Q000737:chemistry / Q000648:ultrastructure; D003110:Colonic Neoplasms / Q000473:pathology* / Q000503:physiopathology* / Q000648:ultrastructure; D017868:Cyclic AMP-Dependent Protein Kinases / Q000494:pharmacology; D004848:Epithelium / Q000737:chemistry / Q000473:pathology / Q000648:ultrastructure; D006801:Humans; D009711:Nucleotides / Q000378:metabolism*; D018408:Patch-Clamp Techniques; D010766:Phosphorylation; D015221:Potassium Channels / Q000032:analysis / Q000502:physiology*; D011493:Protein Kinase C / Q000494:pharmacology; D011494:Protein Kinases / Q000378:metabolism*; D015290:Second Messenger Systems; D014407:Tumor Cells, Cultured
D001427:Bacterial Toxins; D009711:Nucleotides; D015221:Potassium Channels; C036117:staphylotoxin; D000255:Adenosine Triphosphate; D011494:Protein Kinases; D017868:Cyclic AMP-Dependent Protein Kinases; D011493:Protein Kinase C; D002118:Calcium; D015056:1-Methyl-3-isobutylxanthine