Activation of AMPK reduces the co-transporter activity of NKCC1

• Published in: Molecular membrane biology Vol. 31; no. 2-3; pp. 95 - 102
• Main Authors: Fraser, Scott A., Davies, Matthew, Katerelos, Marina, Gleich, Kurt, Choy, Suet-Wan, Steel, Rohan, Galic, Sandra, Mount, Peter F., Kemp, Bruce E., Power, David A.
• Format: Journal Article
• Language: English
• Published: England Informa UK Ltd 01.03.2014; Taylor & Francis
• Subjects: Alanine - metabolism; Aminoimidazole Carboxamide - analogs & derivatives; Aminoimidazole Carboxamide - pharmacology; AMP-Activated Protein Kinases - genetics; AMP-Activated Protein Kinases - metabolism; Animals; Cell biology; Cell Line; Dogs; epithelial cell; Gene Expression Regulation - drug effects; HEK293 Cells; Humans; Madin Darby Canine Kidney Cells; Mice; Na-coupled transport; Phenformin - pharmacology; Phosphorylation; Point Mutation; Protein Transport - drug effects; Pyrones - pharmacology; Ribonucleotides - pharmacology; Serine - metabolism; Solute Carrier Family 12, Member 2 - genetics; Solute Carrier Family 12, Member 2 - metabolism; Thiophenes - pharmacology
• ISSN: 0968-7688; 1464-5203
• DOI: 10.3109/09687688.2014.902128

Abstract 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 NKCC1T212/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.