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.
Human Genetics Unit, Department of Medicine, University of Edinburgh, Molecular Medicine Centre, Western General Hospital, Edinburgh EH4 2XU, UK.
K A
KA
Lansdell
J F
JF
Kidd
S J
SJ
Delaney
B J
BJ
Wainwright
D N
DN
Sheppard
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