The tyrosine kinase p60c-src regulates the fast gate of the cystic fibrosis transmembrane conductance regulator chloride channel.
The role of the tyrosine kinase p60c-src on the gating of the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel was investigated with the cell-attached and excised patch clamp technique in conjunction with current noise analysis of recordings containing multiple channels per patch. Spectra of CFTR-generated current noise contained a low-frequency and a high-frequency Lorentzian noise component. In the cell-attached mode, the high-frequency Lorentzian was significantly dependent on the membrane potential, while the low-frequency Lorentzian was unaffected. Excision of forskolin-stimulated patches into ATP-containing solution significantly reduced the amplitude of the voltage-dependent high-frequency Lorentzian. Addition of the tyrosine kinase p60c-src to excised, active, CFTR-containing membrane patches increased mean currents by 54%, increased the corner frequency of the low-frequency Lorentzian, and recovered the high-frequency Lorentzian and its characteristics. Treatment with lambda-phosphatase inactivated src-induced currents and changes in gating. When active patches were excised under conditions in which patch-associated tyrosine phosphatases were blocked with sodium vanadate, the high-frequency gating remained relatively unchanged. The results suggest that CFTR's open probability and its voltage-dependent fast gate are dependent on tyrosine phosphorylation, and that membrane-associated tyrosine phosphatases are responsible for inactivation of the fast gate after patch excision.
Fischer
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University of California, Department of Molecular and Cell Biology, Berkeley 94720-3200, USA.
Machen
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TE
D016475:3T3 Cells; D000704:Analysis of Variance; D000818:Animals; D002460:Cell Line; D002462:Cell Membrane / Q000502:physiology*; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000502:physiology*; D004848:Epithelium; D006801:Humans; D015640:Ion Channel Gating; D007700:Kinetics; D008564:Membrane Potentials; D051379:Mice; D008956:Models, Chemical; D018408:Patch-Clamp Techniques; D016392:Proto-Oncogene Proteins pp60(c-src) / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D014162:Transfection
C505032:CFTR protein, human; D011994:Recombinant Proteins; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D016392:Proto-Oncogene Proteins pp60(c-src)