Activation of AMPK reduces the co-transporter activity of NKCC1.
The co-transporter activity of Na(+)-K(+)-2Cl(-) 1 (NKCC1) is dependent on phosphorylation. In this study we show the energy-sensing kinase AMPK inhibits NKCC1 activity. Three separate AMPK activators (AICAR, Phenformin and A-769662) inhibited NKCC1 flux in a variety of nucleated cells. Treatment with A-769662 resulted in a reduction of NKCC1(T212/T217) phosphorylation, and this was reversed by treatment with the non-selective AMPK inhibitor Compound C. AMPK dependence was confirmed by treatment of AMPK null mouse embryonic fibroblasts, where A-769662 had no effect on NKCC1 mediated transport. AMPK was found to directly phosphorylate a recombinant human-NKCC1 N-terminal fragment (1-293) with the phosphorylated site identified as S77. Mutation of Serine 77 to Alanine partially prevented the inhibitory effect of A-769662 on NKCC1 activity. In conclusion, AMPK can act to reduce NKCC1-mediated transport. While the exact mechanism is still unclear there is evidence for both a direct effect on phosphorylation of S77 and reduced phosphorylation of T212/217.
Institute for Breathing and Sleep, Kidney Laboratory , Melbourne, Australia .
Scott A
SA
Fraser
Matthew
M
Davies
Marina
M
Katerelos
Kurt
K
Gleich
Suet-Wan
SW
Choy
Rohan
R
Steel
Sandra
S
Galic
Peter F
PF
Mount
Bruce E
BE
Kemp
David A
DA
Power
D055372:AMP-Activated Protein Kinases / Q000235:genetics / Q000378:metabolism*; D000409:Alanine / Q000378:metabolism; D000620:Aminoimidazole Carboxamide / Q000031:analogs & derivatives* / Q000494:pharmacology; D000818:Animals; D001713:Biphenyl Compounds; D002460:Cell Line; D004285:Dogs; D005786:Gene Expression Regulation / Q000187:drug effects; D057809:HEK293 Cells; D006801:Humans; D061985:Madin Darby Canine Kidney Cells; D051379:Mice; D010629:Phenformin / Q000494:pharmacology*; D010766:Phosphorylation; D017354:Point Mutation; D021381:Protein Transport / Q000187:drug effects; D011753:Pyrones / Q000494:pharmacology*; D012265:Ribonucleotides / Q000494:pharmacology*; D012694:Serine / Q000378:metabolism; D064506:Solute Carrier Family 12, Member 2 / Q000235:genetics / Q000378:metabolism*; D013876:Thiophenes / Q000494:pharmacology*
D001713:Biphenyl Compounds; D011753:Pyrones; D012265:Ribonucleotides; D064506:Solute Carrier Family 12, Member 2; D013876:Thiophenes; D000620:Aminoimidazole Carboxamide; D012694:Serine; D010629:Phenformin; D055372:AMP-Activated Protein Kinases; C031143:AICA ribonucleotide; D000409:Alanine; C512408:4-hydroxy-3-(4-(2-hydroxyphenyl)phenyl)-6-oxo-7H-thieno(2,3-b)pyridine-5-carbonitrile