Mechanistic Insight into Control of CFTR by AMPK

Kongsuphol, Patthara; Cassidy, Diane; Hieke, Bernhard; Treharne, Kate J.; Schreiber, Rainer; Mehta, Anil; Kunzelmann, Karl

doi:10.1074/jbc.m806780200

Cited by 75 publications

(101 citation statements)

References 42 publications

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…9, C and D). This observation is different from previous reports based on macroscopic recordings from Xenopus oocytes (24,25) and further suggests that fractional phosphoserine Ser 768 in WT CFTR should be very low when expressed in HEK-293T cells. In vivo single channel recordings further supported this notion.…”

contrasting

confidence: 55%

“…Although most phosphorylation sites, including Ser 700 , Ser 795 , Ser 813 , and Ser 660 , stimulate channel activity, Ser 737 and Ser 768 are inhibitory sites (18). Substitutions of these two residues with alanines increase channel activity (18,19,24,25). In addition, removal of residues 760 -783 or 817-838 (NEG2) or much of the R domain (⌬708 -835/S660A) from CFTR eliminates PKA dependence of channel activity (26 -28).…”

mentioning

confidence: 99%

See 1 more Smart Citation

The Inhibition Mechanism of Non-phosphorylated Ser768 in the Regulatory Domain of Cystic Fibrosis Transmembrane Conductance Regulator

Wang

2011

Journal of Biological Chemistry

View full text Add to dashboard Cite

The cystic fibrosis transmembrane conductance regulator (CFTR) is a member of the ATP-binding cassette transporters but serves as a chloride channel dysfunctional in cystic fibrosis. The activity of CFTR is tightly controlled not only by ATPdriven dimerization of its nucleotide-binding domains but also by phosphorylation of a unique regulatory (R) domain by protein kinase A (PKA of cytoplasmic loop 3 to prevent channel opening by ATP in the non-phosphorylated state and by subsequent cAMP-dependent phosphorylation. These observations support an electron cryomicroscopy-based structural model on which the R domain is closed to cytoplasmic loops regulating channel gating. The cystic fibrosis transmembrane conductance regulator (CFTR)2 chloride channel is widely distributed in the human organs, including the heart, and mediates the electric response to ATP and protein kinase A or C. As shown in Fig. 1, this protein has two membrane-spanning domains (MSD1 and MSD2), two intracellular nucleotide-binding domains (NBD1 and NBD2), and a unique regulatory (R) domain, although it belongs to the human C subfamily of ATP-binding cassette transporters (1, 2). Each MSD consists of six transmembrane helical segments probably extended to four cytoplasmic loops (3). Although recent studies have strongly suggested structural similarities between CFTR and bacterial transporters Sav1866 and MsbA (3-5), three-dimensional structural information about the whole protein is still unavailable except for the crystal structure of the isolated NBD1 (6). Furthermore, the exact location and relative orientation of the R domain in the whole protein are also unclear because this domain lacks a stably folded globular structure and thus is disordered (7,8).Ion transport of CFTR is triggered by not only ATP binding and hydrolysis at the interface of a NBD1-NBD2 dimer but also phosphorylation by protein kinase A (PKA) (9). Structures of bacterial NBD homodimers indicate two ATP-binding sites at the NBD1-NBD2 interface, and each site is composed of residues from both NBDs (10). However, most PKA phosphorylation sites are mainly found in the R domain (6, 11).CFTR activity is tightly controlled by interdomain interactions. Several thiol-specific cross-linking studies, based on the crystal structures of Sav1866 and MsbA, have shown that the NBD1-NBD2 dimerization drives channel opening (12). However, chemical cross-linking of NBDs to cytoplasmic loops (CLs) inhibits channel activity (Fig. 1) (5, 13, 14). Recent structural studies of CFTR and other ATP-binding cassette transporters suggested rearrangements of CLs that couple dimerization of the NBDs to a change in the MSDs from an inward to an outward facing conformation (4,15,16). Our recent study also demonstrated that a K190C/S mutation from CL1 enhances ATP-independent channel opening induced by a K978C/P/S mutation from CL3 (17). Thus, CLs may function as a key regulatory switch to modulate normal CFTR activity. * This work was supported, in whole or in part, by National Institutes of Health Grant 2R...

show abstract

contrasting

confidence: 55%

mentioning

confidence: 99%

The Inhibition Mechanism of Non-phosphorylated Ser768 in the Regulatory Domain of Cystic Fibrosis Transmembrane Conductance Regulator

Wang

2011

Journal of Biological Chemistry

View full text Add to dashboard Cite

show abstract

“…It has multiple phosphorylation sites, most of which are stimulatory except Ser-768 and Ser-737 (9,10,12,(15)(16)(17)(18). Recent functional studies revealed that NEG2 (1B), which is a small C-terminal segment (817-838) of the R domain and has a conserved helical region and a net charge of Ϫ9, plays a critical role in CFTR gating and trafficking in response to cAMP stimulation (19,20).…”

mentioning

confidence: 99%

Regulation of Activation and Processing of the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) by a Complex Electrostatic Interaction between the Regulatory Domain and Cytoplasmic Loop 3

Wang

Duan

2012

Journal of Biological Chemistry

View full text Add to dashboard Cite

Background: NEG2 regulates CFTR gating but the mechanism is unknown. Results: A putative NEG2-CL3 electrostatic attraction, possibly weakened by Arg-764/Arg-766 of the R domain, prohibited CFTR activation. A charge exchange between NEG2 and CL3 caused misprocessing. Conclusion: Electrostatic regulation of CFTR activation and processing may be asymmetric at the CL3-R interface. Significance: The CL3-R interface is optimally designed for multiple regulations of CFTR functions.

show abstract

“…A number of in vitro and in vivo studies have shown that activation of AMPK inhibits PKA-activated CFTR activity via a mechanism involving AMPK-mediated phosphorylation at inhibitory sites within CFTR regulatory domain. 12) Our experiments in T84 cells demonstrated that the inhibitory effect of both high and low doses of DHIS on CFTR-mediated apical Cl − current was markedly reduced by pretreatment with compound C, a specific aMPK inhibitor (Fig. 4).…”

Section: Discussionmentioning

confidence: 93%

“…[8][9][10][11] Recent studies demonstrated that AMPK phosphorylates at serine 737 and serine 768 in the R domain, resulting in inhibition of both basal and PKA-stimulated CFTR activities. 12) Stevioside is a natural sweetener extracted from Stevia rebaudiana. Stevioside has been shown to possess a number of health benefits including anti-hypertension, anti-hyperglycemia, anti-tumor and anti-inflammation.…”

Section: Cystic Fibrosis Transmembrane Conductance Regulator (Cftr) Imentioning

confidence: 99%

Activation of AMP-Activated Protein Kinase by a Plant-Derived Dihydroisosteviol in Human Intestinal Epithelial Cell

Muanprasat

Sirianant

Sawasvirojwong

et al. 2013

Biological & Pharmaceutical Bulletin

View full text Add to dashboard Cite

Our previous study has shown that dihydroisosteviol (DHIS), a derivative of stevioside isolated from Stevia rebaudiana (Bertoni), inhibits cystic fibrosis transmembrane conductance regulator (CFTR)-mediated transepithelial chloride secretion across monolayers of human intestinal epithelial (T84) cells and prevents cholera toxin-induced intestinal fluid secretion in mouse closed loop models. In this study, we aimed to investigate a mechanism by which DHIS inhibits CFTR activity. Apical chloride current measurements in Fisher rat thyroid cells stably transfected with wild-type human CFTR (FRT-CFTR cells) and T84 cells were used to investigate mechanism of CFTR inhibition by DHIS. In addition, effect of DHIS on AMP-activated protein kinase (AMPK) activation was investigated using Western blot analysis. Surprisingly, it was found that DHIS failed to inhibit CFTR-mediated apical chloride current in FRT-CFTR cells. In contrast, DHIS effectively inhibited CFTR-mediated apical chloride current induced by a cell permeable cAMP analog CPT-cAMP and a direct CFTR activator genistein in T84 cell monolayers. Interestingly, this inhibitory effect of DHIS on CFTR was significantly (p<0.05) reduced by pretreatment with compound C, an AMPK inhibitor. AICAR, a known AMPK activator, was able to inhibit CFTR activity in both FRT-CFTR and T84 cells. Western blot analysis showed that DHIS induced AMPK activation in T84 cells, but not in FRT-CFTR cells. Our results indicate that DHIS inhibits CFTR-mediated chloride secretion in T84 cells, in part, by activation of AMPK activity. DHIS therefore represents a novel candidate of AMPK activators.

show abstract

Mechanistic Insight into Control of CFTR by AMPK

Cited by 75 publications

References 42 publications

The Inhibition Mechanism of Non-phosphorylated Ser768 in the Regulatory Domain of Cystic Fibrosis Transmembrane Conductance Regulator

The Inhibition Mechanism of Non-phosphorylated Ser768 in the Regulatory Domain of Cystic Fibrosis Transmembrane Conductance Regulator

Regulation of Activation and Processing of the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) by a Complex Electrostatic Interaction between the Regulatory Domain and Cytoplasmic Loop 3

Activation of AMP-Activated Protein Kinase by a Plant-Derived Dihydroisosteviol in Human Intestinal Epithelial Cell

Contact Info

Product

Resources

About