Regulation of murine cystic fibrosis transmembrane conductance regulator Cl- channels expressed in Chinese hamster ovary cells.
1. We investigated the effect of protein kinases and phosphatases on murine cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channels, expressed in Chinese hamster ovary (CHO) cells, using iodide efflux and the excised inside-out configuration of the patch-clamp technique. 2. The protein kinase C (PKC) activator, phorbol dibutyrate, enhanced cAMP-stimulated iodide efflux. However, PKC did not augment the single-channel activity of either human or murine CFTR Cl- channels that had previously been activated by protein kinase A. 3. Fluoride, a non-specific inhibitor of protein phosphatases, stimulated both human and murine CFTR Cl- channels. However, calyculin A, a potent inhibitor of protein phosphatases 1 and 2A, did not enhance cAMP-stimulated iodide efflux. 4. The alkaline phosphatase inhibitor, (-)-bromotetramisole augmented cAMP-stimulated iodide efflux and, by itself, stimulated a larger efflux than that evoked by cAMP agonists. However, (+)-bromotetramisole, the inactive enantiomer, had the same effect. For murine CFTR, neither enantiomer enhanced single-channel activity. In contrast, both enantiomers increased the open probability (Po) of human CFTR, suggesting that bromotetramisole may promote the opening of human CFTR. 5. As murine CFTR had a low Po and was refractory to stimulation by activators of human CFTR, we investigated whether murine CFTR may open to a subconductance state. When single-channel records were filtered at 50 Hz, a very small subconductance state of murine CFTR was observed that had a Po greater than that of human CFTR. The occupancy of this subconductance state may explain the differences in channel regulation observed between human and murine CFTR.
Lansdell
K A
KA
Human Genetics Unit, Department of Medicine, University of Edinburgh, Molecular Medicine Centre, Western General Hospital, Edinburgh EH4 2XU, UK.
Kidd
J F
JF
Delaney
S J
SJ
Wainwright
B J
BJ
Sheppard
D N
DN
D000465:Algorithms; D000818:Animals; D016466:CHO Cells; D018118:Chloride Channels / Q000378:metabolism*; D006224:Cricetinae; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000378:metabolism*; D004558:Electric Stimulation; D004594:Electrophysiology; D004789:Enzyme Activation / Q000187:drug effects / Q000502:physiology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005459:Fluorides / Q000494:pharmacology; D006801:Humans; D007454:Iodides / Q000378:metabolism; D008564:Membrane Potentials / Q000502:physiology; D051379:Mice; D018408:Patch-Clamp Techniques; D010749:Phosphoprotein Phosphatases / Q000037:antagonists & inhibitors / Q000494:pharmacology; D011493:Protein Kinase C / Q000378:metabolism; D047428:Protein Kinase Inhibitors; D011494:Protein Kinases / Q000494:pharmacology
C505032:CFTR protein, human; D018118:Chloride Channels; D004791:Enzyme Inhibitors; D007454:Iodides; D047428:Protein Kinase Inhibitors; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D011494:Protein Kinases; D011493:Protein Kinase C; D010749:Phosphoprotein Phosphatases; D005459:Fluorides