AMP-activated protein kinase inhibits KCNQ1 channels through regulation of the ubiquitin ligase Nedd4-2 in renal epithelial cells

The KCNQ1 K+ channel plays a key role in the regulation of several physiological functions, including cardiac excitability, cardiovascular tone, and body electrolyte homeostasis. The metabolic sensor AMP-activated protein kinase (AMPK) has been shown to regulate a growing number of ion transport proteins. To determine whether AMPK regulates KCNQ1, we studied the effects of AMPK activation on KCNQ1 currents in Xenopus laevis oocytes and collecting duct epithelial cells. AMPK activation decreased KCNQ1 currents and channel surface expression in X. laevis oocytes, but AMPK did not phosphorylate KCNQ1 in vitro, suggesting an indirect regulatory mechanism. As it has been recently shown that the ubiquitin-protein ligase Nedd4-2 inhibits KCNQ1 plasma membrane expression and that AMPK regulates epithelial Na+ channels via Nedd4-2, we examined the role of Nedd4-2 in the AMPK-dependent regulation of KCNQ1. Channel inhibition by AMPK was blocked in oocytes coexpressing either a dominant-negative or constitutively active Nedd4-2 mutant, or a Nedd4-2 interaction-deficient KCNQ1 mutant, suggesting that Nedd4-2 participates in the regulation of KCNQ1 by AMPK. KCNQ1 is expressed at the basolateral membrane in mouse polarized kidney cortical collecting duct (mpkCCDc14) cells and in rat kidney. Treatment with the AMPK activators AICAR (2 mM) or metformin (1 mM) reduced basolateral KCNQ1 currents in apically permeabilized polarized mpkCCDc14 cells. Moreover, AICAR treatment of rat kidney slices ex vivo induced AMPK activation and intracellular redistribution of KCNQ1 from the basolateral membrane in collecting duct principal cells. AICAR treatment also induced increased ubiquitination of KCNQ1 immunoprecipitated from kidney slice homogenates. These results indicate that AMPK inhibits KCNQ1 activity by promoting Nedd4-2-dependent channel ubiquitination and retrieval from the plasma membrane.

Download Full-text

α1-AMP-Activated Protein Kinase Regulates Hypoxia-Induced Na,K-ATPase Endocytosis via Direct Phosphorylation of Protein Kinase Cζ

Molecular and Cellular Biology ◽

10.1128/mcb.00054-09 ◽

2009 ◽

Vol 29 (13) ◽

pp. 3455-3464 ◽

Cited By ~ 75

Author(s):

Galina A. Gusarova ◽

Laura A. Dada ◽

Aileen M. Kelly ◽

Chaya Brodie ◽

Lee A. Witters ◽

...

Keyword(s):

Protein Kinase ◽

Epithelial Cells ◽

A549 Cells ◽

Dominant Negative ◽

Alveolar Epithelial Cells ◽

Ampk Activation ◽

Α Subunit ◽

Alveolar Epithelial ◽

Mitochondrial Ros ◽

Amp Activated Protein Kinase

ABSTRACT Hypoxia promotes Na,K-ATPase endocytosis via protein kinase Cζ (PKCζ)-mediated phosphorylation of the Na,K-ATPase α subunit. Here, we report that hypoxia leads to the phosphorylation of 5′-AMP-activated protein kinase (AMPK) at Thr172 in rat alveolar epithelial cells. The overexpression of a dominant-negative AMPK α subunit (AMPK-DN) construct prevented the hypoxia-induced endocytosis of Na,K-ATPase. The overexpression of the reactive oxygen species (ROS) scavenger catalase prevented hypoxia-induced AMPK activation. Moreover, hypoxia failed to activate AMPK in mitochondrion-deficient ρ0-A549 cells, suggesting that mitochondrial ROS play an essential role in hypoxia-induced AMPK activation. Hypoxia-induced PKCζ translocation to the plasma membrane and phosphorylation at Thr410 were prevented by the pharmacological inhibition of AMPK or by the overexpression of the AMPK-DN construct. We found that AMPK α phosphorylates PKCζ on residue Thr410 within the PKCζ activation loop. Importantly, the activation of AMPK α was necessary for hypoxia-induced AMPK-PKCζ binding in alveolar epithelial cells. The overexpression of T410A mutant PKCζ prevented hypoxia-induced Na,K-ATPase endocytosis, confirming that PKCζ Thr410 phosphorylation is essential for this process. PKCζ activation by AMPK is isoform specific, as small interfering RNA targeting the α1 but not the α2 catalytic subunit prevented PKCζ activation. Accordingly, we provide the first evidence that hypoxia-generated mitochondrial ROS lead to the activation of the AMPK α1 isoform, which binds and directly phosphorylates PKCζ at Thr410, thereby promoting Na,K-ATPase endocytosis.

Download Full-text

Epithelial Sodium Channel Inhibition by AMP-activated Protein Kinase in Oocytes and Polarized Renal Epithelial Cells

Journal of Biological Chemistry ◽

10.1074/jbc.m501770200 ◽

2005 ◽

Vol 280 (18) ◽

pp. 17608-17616 ◽

Cited By ~ 111

Author(s):

Marcelo D. Carattino ◽

Robert S. Edinger ◽

Heather J. Grieser ◽

Rosalee Wise ◽

Dietbert Neumann ◽

...

Keyword(s):

Protein Kinase ◽

Epithelial Cells ◽

Sodium Channel ◽

Epithelial Sodium Channel ◽

Renal Epithelial Cells ◽

Channel Inhibition ◽

Amp Activated Protein Kinase

Download Full-text

Forskolin stimulates phosphorylation and membrane accumulation of UT-A3

AJP Renal Physiology ◽

10.1152/ajprenal.00197.2007 ◽

2007 ◽

Vol 293 (4) ◽

pp. F1308-F1313 ◽

Cited By ~ 64

Author(s):

Mitsi A. Blount ◽

Janet D. Klein ◽

Christopher F. Martin ◽

Dmitry Tchapyjnikov ◽

Jeff M. Sands

Keyword(s):

Plasma Membrane ◽

Plasma Membranes ◽

Apical Membrane ◽

Collecting Duct ◽

Rat Kidney ◽

Basolateral Membrane ◽

Apical Plasma Membrane ◽

Membrane Staining ◽

Protein Protein Interaction ◽

Medullary Collecting Duct

UT-A1 is regulated by vasopressin and is localized to the apical membrane and intracellular compartment of inner medullary collecting duct (IMCD) cells. UT-A3 is also expressed in the IMCD and is regulated by forskolin in heterologous systems. The goal of the present study is to investigate mechanisms by which vasopressin regulates UT-A3 in rat IMCD. In fresh suspensions of rat IMCD, forskolin increases the phosphorylation of UT-A3, similar to UT-A1. Biotinylation studies indicate that UT-A3 is located in the plasma membrane. Forskolin treatment increases the abundance of UT-A3 in the plasma membrane similar to UT-A1. However, these two transporters do not form a complex through a protein-protein interaction, suggesting that transporter function is unique to each protein. While immunohistochemistry localized UT-A3 to the basal and lateral membranes, a majority of the staining was cytosolic. Immunohistochemistry of vasopressin-treated rat kidney sections also localized UT-A3 primarily to the cytosol with basal and lateral membrane staining but also showed some apical membrane staining in some IMCD cells. This suggests that under normal conditions, UT-A3 functions as the basolateral transporter but in a high cAMP environment, the transporter may move from the cytosol to all plasma membranes to increase urea flux in the IMCD. In summary, this study confirms that UT-A3 is located in the inner medullary tip where it is expressed in the basolateral membrane, shows that UT-A3 is a phosphoprotein in rat IMCD that can be trafficked to the plasma membrane independent of UT-A1, and suggests that vasopressin may induce UT-A3 expression in the apical plasma membrane of IMCD.

Download Full-text

AMP-activated Protein Kinase (AMPK) Activation and Glycogen Synthase Kinase-3β (GSK-3β) Inhibition Induce Ca2+-independent Deposition of Tight Junction Components at the Plasma Membrane

Journal of Biological Chemistry ◽

10.1074/jbc.m110.186932 ◽

2011 ◽

Vol 286 (19) ◽

pp. 16879-16890 ◽

Cited By ~ 41

Author(s):

Li Zhang ◽

Francois Jouret ◽

Jesse Rinehart ◽

Jeff Sfakianos ◽

Ira Mellman ◽

...

Keyword(s):

Plasma Membrane ◽

Protein Kinase ◽

Tight Junction ◽

Glycogen Synthase ◽

Glycogen Synthase Kinase ◽

Ampk Activation ◽

Glycogen Synthase Kinase 3Β ◽

Gsk 3Β ◽

Amp Activated Protein Kinase

Download Full-text

Molecular characterization of a novel urea transporter from kidney inner medullary collecting ducts

AJP Renal Physiology ◽

10.1152/ajprenal.2001.280.3.f487 ◽

2001 ◽

Vol 280 (3) ◽

pp. F487-F494 ◽

Cited By ~ 26

Author(s):

Chairat Shayakul ◽

Hiroyasu Tsukaguchi ◽

Urs V. Berger ◽

Matthias A. Hediger

Keyword(s):

Amino Acid ◽

Molecular Characterization ◽

Collecting Duct ◽

Rat Kidney ◽

Basolateral Membrane ◽

Functional Characterization ◽

Xenopus Laevis Oocytes ◽

Urea Transporter ◽

Urea Uptake

In the terminal part of the kidney collecting duct, rapid urea reabsorption is essential to maintaining medullary hypertonicity, allowing maximal urinary concentration to occur. This process is mediated by facilitated urea transporters on both apical and basolateral membranes. Our previous studies have identified three rat urea transporters involved in the urinary concentrating mechanism, UT1, UT2 and UT3 , herein renamed UrT1-A, UrT1-B, and UrT2, which exhibit distinct spatial distribution in the kidney. Here we report the molecular characterization of an additional urea transporter isoform, UrT1-C, from rat kidney that encodes a 460-amino acid residue protein. UrT1-C has 70 and 62% amino acid identity to rat UrT1-B and UrT2 (UT3), respectively, and 99% identity to a recently reported rat isoform (UT-A3; Karakashian A, Timmer RT, Klein JD, Gunn RB, Sands JM, and Bagnasco SM. J Am Soc Nephrol 10: 230–237, 1999). We report the anatomic distribution of UrT1-C in the rat kidney tubule system as well as a detailed functional characterization. UrT1-C m RNA is primarily expressed in the deep part of the inner medulla. When expressed in Xenopus laevis oocytes, UrT1-C induced a 15-fold stimulation of urea uptake, which was inhibited almost completely by phloretin (0.7 mM) and 60–95% by thiourea analogs (150 mM). The characteristics are consistent with those described in perfusion studies with inner medullary collecting duct (IMCD) segments, but, contrary to UrT1-A, UrT1-C-mediated urea uptake was not stimulated by activation of protein kinase A. Our data show that UrT1-C is a phloretin-inhibitable urea transporter expressed in the terminal collecting duct that likely serves as an exit mechanism for urea at the basolateral membrane of IMCD cells.

Download Full-text

Adaptor protein 1 complexes regulate intracellular trafficking of the kidney anion exchanger 1 in epithelial cells

AJP Cell Physiology ◽

10.1152/ajpcell.00124.2012 ◽

2012 ◽

Vol 303 (5) ◽

pp. C554-C566 ◽

Cited By ~ 16

Author(s):

Ensaf Y. Almomani ◽

Jennifer C. King ◽

Janjuree Netsawang ◽

Pa-Thai Yenchitsomanus ◽

Prida Malasit ◽

...

Keyword(s):

Plasma Membrane ◽

Epithelial Cells ◽

Anion Exchanger ◽

Collecting Duct ◽

Basolateral Membrane ◽

Adaptor Protein ◽

Cortical Collecting Duct ◽

Anion Exchanger 1 ◽

Recycling Endosomes ◽

Carboxyl Terminal

Distal renal tubular acidosis (dRTA) can be caused by mutations in the gene encoding the anion exchanger 1 (AE1) and is characterized by defective urinary acidification, metabolic acidosis, and renal stones. AE1 is expressed at the basolateral membrane of type A intercalated cells in the renal cortical collecting duct (kAE1). Two dRTA mutations result in the carboxyl-terminal truncation of kAE1; in one case, the protein trafficked in a nonpolarized way in epithelial cells. A recent yeast two-hybrid assay showed that the carboxyl-terminal cytosolic domain of AE1 interacts with adaptor protein complex 1 (AP-1A) subunit μ1A (mu-1A; Sawasdee N, Junking M, Ngaojanlar P, Sukomon N, Ungsupravate D, Limjindaporn T, Akkarapatumwong V, Noisakran S, Yenchitsomanus PT. Biochem Biophys Res Commun 401: 85–91, 2010). Here, we show the interaction between kAE1 and mu-1A and B in vitro by reciprocal coimmunoprecipitation in epithelial cells and in vivo by coimmunoprecipitation from mouse kidney extract. When endogenous mu-1A (and to a lesser extent mu-1B) was reduced, kAE1 protein was unable to traffic to the plasma membrane and was rapidly degraded via a lysosomal pathway. Expression of either small interfering RNA-resistant mu-1A or mu-1B stabilized kAE1 in these cells. We also show that newly synthesized kAE1 does not traffic through recycling endosomes to the plasma membrane, suggesting that AP-1B, located in recycling endosomes, is not primarily involved in trafficking of newly synthesized kAE1 when AP-1A is present in the cells. Our data demonstrate that AP-1A regulates processing of the basolateral, polytopic membrane protein kAE1 to the cell surface and that both AP-1A and B adaptor complexes are required for normal kAE1 trafficking.

Download Full-text

Inhibition of the KCa3.1 channels by AMP-activated protein kinase in human airway epithelial cells

AJP Cell Physiology ◽

10.1152/ajpcell.00418.2008 ◽

2009 ◽

Vol 296 (2) ◽

pp. C285-C295 ◽

Cited By ~ 44

Author(s):

Hélène Klein ◽

Line Garneau ◽

Nguyen Thu Ngan Trinh ◽

Anik Privé ◽

François Dionne ◽

...

Keyword(s):

Protein Kinase ◽

Epithelial Cells ◽

Short Circuit ◽

Basolateral Membrane ◽

Airway Epithelial Cells ◽

Functional Link ◽

Compound C ◽

Amp Activated Protein Kinase ◽

Inside Out ◽

Two Hybrid

The vectorial transport of ions and water across epithelial cells depends to a large extent on the coordination of the apical and basolateral ion fluxes with energy supply. In this work we provide the first evidence for a regulation by the 5′-AMP-activated protein kinase (AMPK) of the calcium-activated potassium channel KCa3.1 expressed at the basolateral membrane of a large variety of epithelial cells. Inside-out patch-clamp experiments performed on human embryonic kidney (HEK) cells stably transfected with KCa3.1 first revealed a decrease in KCa3.1 activity following the internal addition of AMP at a fixed ATP concentration. This effect was dose dependent with half inhibition at 140 μM AMP in 1 mM ATP. Evidence for an interaction between the COOH-terminal region of KCa3.1 and the γ1-subunit of AMPK was next obtained by two-hybrid screening and pull-down experiments. Our two-hybrid analysis confirmed in addition that the amino acids extending from Asp380 to Ala400 in COOH-terminal were essential for the interaction AMPK-γ1/KCa3.1. Inside-out experiments on cells coexpressing KCa3.1 with the dominant negative AMPK-γ1-R299G mutant showed a reduced sensitivity of KCa3.1 to AMP, arguing for a functional link between KCa3.1 and the γ1-subunit of AMPK. More importantly, coimmunoprecipitation experiments carried out on bronchial epithelial NuLi cells provided direct evidence for the formation of a KCa3.1/AMPK-γ1 complex at endogenous AMPK and KCa3.1 expression levels. Finally, treating NuLi monolayers with the membrane permeant AMPK activator 5-aminoimidazole-4-carboxamide-1-β-d-ribofuranoside (AICAR) caused a significant decrease of the KCa3.1-mediated short-circuit currents, an effect reversible by coincubation with the AMPK inhibitor Compound C. These observations argue for a regulation of KCa3.1 by AMPK in a functional epithelium through protein/protein interactions involving the γ1-subunit of AMPK.

Download Full-text