Mechanistic insight into control of CFTR by AMPK.
The cystic fibrosis transmembrane conductance regulator (CFTR) is a cAMP and protein kinase A (PKA)-regulated Cl(-) channel in the apical membrane of epithelial cells. The metabolically regulated and adenosine monophosphate-stimulated kinase (AMPK) is colocalized with CFTR and attenuates its function. However, the sites for CFTR phosphorylation and the precise mechanism of inhibition of CFTR by AMPK remain obscure. We demonstrate that CFTR normally remains closed at baseline, but nevertheless, opens after inhibition of AMPK. AMPK phosphorylates CFTR in vitro at two essential serines (Ser(737) and Ser(768)) in the R domain, formerly identified as "inhibitory" PKA sites. Replacement of both serines by alanines (i) reduced phosphorylation of the R domain, with Ser(768) having dramatically greater impact, (ii) produced CFTR channels that were partially open in the absence of any stimulation, (iii) significantly augmented their activation by IBMX/forskolin, and (iv) eliminated CFTR inhibition post AMPK activation. Attenuation of CFTR by AMPK activation was detectable in the absence of cAMP-dependent stimulation but disappeared in maximally stimulated oocytes. Our data also suggest that AMP is produced by local phosphodiesterases in close proximity to CFTR. Thus we propose that CFTR channels are kept closed in nonstimulated epithelia with high baseline AMPK activity but CFTR may be basally active in tissues with lowered endogenous AMPK activity.
Kongsuphol
Patthara
P
Department of Physiology, University of Regensburg, 93053 Regensburg, Germany.
Cassidy
Diane
D
Hieke
Bernhard
B
Treharne
Kate J
KJ
Schreiber
Rainer
R
Mehta
Anil
A
Kunzelmann
Karl
K
D055372:AMP-Activated Protein Kinases / Q000378:metabolism*; D000818:Animals; D002478:Cells, Cultured; D018118:Chloride Channels / Q000502:physiology; D000242:Cyclic AMP / Q000494:pharmacology; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000235:genetics / Q000378:metabolism*; D004847:Epithelial Cells / Q000187:drug effects / Q000378:metabolism; D005455:Fluorescent Antibody Technique; D006801:Humans; D015640:Ion Channel Gating / Q000502:physiology*; D009079:Mucociliary Clearance; D009297:Nasal Mucosa / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D010766:Phosphorylation
C505032:CFTR protein, human; D018118:Chloride Channels; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D000242:Cyclic AMP; D055372:AMP-Activated Protein Kinases