Regulation of Na+-K+-Cl− cotransporter type 2 by the with no lysine kinase-dependent signaling pathway

2019 ◽  
Vol 317 (1) ◽  
pp. C20-C30 ◽  
Author(s):  
Andrée-Anne Marcoux ◽  
Samira Slimani ◽  
Laurence E. Tremblay ◽  
Rachelle Frenette-Cotton ◽  
Alexandre P. Garneau ◽  
...  
Keyword(s):  
Cell Surface ◽  
Ion Transport ◽  
Transmembrane Domain ◽  
Expression System ◽  
Xenopus Laevis Oocyte ◽  
Thick Ascending Limb ◽  
Cell Shrinkage ◽  
Enzymatic Function ◽  
Wnk Kinase

Na+-K+-Cl− cotransporter type 2 (NKCC2) is confined to the apical membrane of the thick ascending limb of Henle, where it reabsorbs a substantial fraction of the ultrafiltered NaCl load. It is expressed along this nephron segment as three main splice variants (called NKCC2A, NKCC2B, and NKCC2F) that differ in residue composition along their second transmembrane domain and first intracellular cytosolic connecting segment (CS2). NKCC2 is known to be activated by cell shrinkage and intracellular [Cl−] reduction. Although the with no lysine (WNK) kinases could play a role in this response, the mechanisms involved are ill defined, and the possibility of variant-specific responses has not been tested thus far. In this study, we have used the Xenopus laevis oocyte expression system to gain further insight in these regards. We have found for the first time that cell shrinkage could stimulate NKCC2A- and NKCC2B-mediated ion transport by increasing carrier abundance at the cell surface and that this response was achieved (at least in part) by the enzymatic function of a WNK kinase. Interestingly, we have also found that the activity and cell surface abundance of NKCC2F were less affected by cell shrinkage compared with the other variants and that ion transport by certain variants could be stimulated through WNK kinase expression in the absence of carrier redistribution. Taken together, these results suggest that the WNK kinase-dependent pathway can affect both the trafficking as well as intrinsic activity of NKCC2 and that CS2 plays an important role in carrier regulation.

scholarly journals Down-Regulation of Cell Surface Receptors Is Modulated by Polar Residues within the Transmembrane Domain

2000 ◽  
Vol 11 (8) ◽  
pp. 2643-2655 ◽  
Author(s):  
Lolita Zaliauskiene ◽  
Sunghyun Kang ◽  
Christie G. Brouillette ◽  
Jacob Lebowitz ◽  
Ramin B. Arani ◽  
...  
Keyword(s):  
Cell Surface ◽  
Transmembrane Domain ◽  
Gradient Centrifugation ◽  
Low Density Lipoprotein ◽  
Density Lipoprotein ◽  
Surface Proteins ◽  
Down Regulation ◽  
Surface Receptors ◽  
Receptor Complexes ◽  
Polar Residues

How recycling receptors are segregated from down-regulated receptors in the endosome is unknown. In previous studies, we demonstrated that substitutions in the transferrin receptor (TR) transmembrane domain (TM) convert the protein from an efficiently recycling receptor to one that is rapidly down regulated. In this study, we demonstrate that the “signal” within the TM necessary and sufficient for down-regulation is Thr11Gln17Thr19 (numbering in TM). Transplantation of these polar residues into the wild-type TR promotes receptor down-regulation that can be demonstrated by changes in protein half-life and in receptor recycling. Surprisingly, this modification dramatically increases the TR internalization rate as well (∼79% increase). Sucrose gradient centrifugation and cross-linking studies reveal that propensity of the receptors to self-associate correlates with down-regulation. Interestingly, a number of cell surface proteins that contain TM polar residues are known to be efficiently down-regulated, whereas recycling receptors for low-density lipoprotein and transferrin conspicuously lack these residues. Our data, therefore, suggest a simple model in which specific residues within the TM sequences dramatically influence the fate of membrane proteins after endocytosis, providing an alternative signal for down-regulation of receptor complexes to the well-characterized cytoplasmic tail targeting signals.

scholarly journals Pleiotropic functions of the transmembrane domain 6 of human melanocortin-4 receptor

2012 ◽  
Vol 49 (3) ◽  
pp. 237-248 ◽  
Author(s):  
Hui Huang ◽  
Ya-Xiong Tao
Keyword(s):  
Cell Surface ◽  
Structure Function ◽  
Transmembrane Domain ◽  
Mapk Pathway ◽  
Surface Expression ◽  
Camp Signaling ◽  
Cell Surface Expression ◽  
Melanocortin 4 Receptor ◽  
Function Relationship ◽  
Relationship Of

The melanocortin-4 receptor (MC4R) is a critical regulator of energy homeostasis and has emerged as a premier target for obesity treatment. Numerous mutations in transmembrane domain 6 (TM6) of MC4R resulting in functional alterations have been identified in obese patients. Several mutagenesis studies also provided some data suggesting the importance of this domain in receptor function. To gain a better understanding of the structure–function relationship of the receptor, we performed alanine-scanning mutagenesis in TM6 to determine the functions of side chains. Of the 31 residues, two were important for cell surface expression, five were indispensable for α-melanocyte-stimulating hormone (α-MSH) and β-MSH binding, and six were important for signaling in the Gs–cAMP–PKA pathway. H264A, targeted normally to the plasma membrane, was undetectable by competitive binding assay and severely defective in basal and stimulated cAMP production and ERK1/2 phosphorylation. Nine mutants had decreased basal cAMP signaling. Seven mutants were constitutively active in cAMP signaling and their basal activities could be inhibited by two MC4R inverse agonists, Ipsen 5i and ML00253764. Five mutants were also constitutively active in the MAPK pathway with enhanced basal ERK1/2 phosphorylation. In summary, our study provided comprehensive data on the structure–function relationship of the TM6 of MC4R. We identified residues that are important for cell surface expression, ligand binding, cAMP generation, and residues for maintaining the WT receptor in active conformation. We also reported constitutive activation of the MAPK pathway and biased signaling. These data will be useful for rationally designing MC4R agonists and antagonists for treatment of eating disorders.

scholarly journals Modulation of cardiac Na+,K+-ATPase cell surface abundance by simulated ischemia-reperfusion and ouabain preconditioning

2013 ◽  
Vol 304 (1) ◽  
pp. H94-H103 ◽  
Author(s):  
Aude Belliard ◽  
Yoann Sottejeau ◽  
Qiming Duan ◽  
Jessa L. Karabin ◽  
Sandrine V. Pierre
Keyword(s):  
Cell Surface ◽  
Atpase Activity ◽  
Ion Transport ◽  
Cell Viability ◽  
Ischemia Reperfusion ◽  
Resistant Mutant ◽  
Surface Expression ◽  
Live Cells ◽  
Mutant Form ◽  
Dependent Manner

Na+,K+-ATPase and cell survival were investigated in a cellular model of ischemia-reperfusion (I/R)-induced injury and protection by ouabain-induced preconditioning (OPC). Rat neonatal cardiac myocytes were subjected to 30 min of substrate and coverslip-induced ischemia followed by 30 min of simulated reperfusion. This significantly compromised cell viability as documented by lactate dehydrogenase release and Annexin V/propidium iodide staining. Total Na+,K+-ATPase α1- and α3-polypeptide expression remained unchanged, but cell surface biotinylation and immunostaining studies revealed that α1-cell surface abundance was significantly decreased. Na+,K+-ATPase-activity in crude homogenates and 86Rb+ transport in live cells were both significantly decreased by about 30% after I/R. OPC, induced by a 4-min exposure to 10 μM ouabain that ended 8 min before the beginning of ischemia, increased cell viability in a PKCε-dependent manner. This was comparable with the protective effect of OPC previously reported in intact heart preparations. OPC prevented I/R-induced decrease of Na+,K+-ATPase activity and surface expression. This model also revealed that Na+,K+-ATPase-mediated 86Rb+ uptake was not restored to control levels in the OPC group, suggesting that the increased viability was not conferred by an increased Na+,K+-ATPase-mediated ion transport capacity at the cell membrane. Consistent with this observation, transient expression of an internalization-resistant mutant form of Na+,K+-ATPase α1 known to have increased surface abundance without increased ion transport activity successfully reduced I/R-induced cell death. These results suggest that maintenance of Na+,K+-ATPase cell surface abundance is critical to myocyte survival after an ischemic attack and plays a role in OPC-induced protection. They further suggest that the protection conferred by increased surface expression of Na+,K+-ATPase may be independent of ion transport.

scholarly journals Characterization of a cDNA encoding a cysteine-rich cell surface protein located in the flagellar pocket of the protozoan Trypanosoma brucei.

1990 ◽  
Vol 10 (9) ◽  
pp. 4506-4517 ◽  
Author(s):  
M G Lee ◽  
B E Bihain ◽  
D G Russell ◽  
R J Deckelbaum ◽  
L H Van der Ploeg
Keyword(s):  
Amino Acid ◽  
Cell Surface ◽  
Trypanosoma Brucei ◽  
Transmembrane Domain ◽  
Density Lipoprotein ◽  
Surface Protein ◽  
Integral Membrane Protein ◽  
Cell Surface Receptor ◽  
Flagellar Pocket ◽  
Cytoplasmic Extension

We have characterized a cDNA encoding a cysteine-rich, acidic integral membrane protein (CRAM) of the parasitic protozoa Trypanosoma brucei and Trypanosoma equiperdum. Unlike other membrane proteins of T. brucei, which are distributed throughout the cell surface, CRAM is concentrated in the flagellar pocket, an invagination of the cell surface of the trypanosome where endocytosis has been documented. Accordingly, CRAM also locates to vesicles located underneath the pocket, providing evidence of its internalization. CRAM has a predicted molecular mass of 130 kilodaltons and has a signal peptide, a transmembrane domain, and a 41-amino-acid cytoplasmic extension. A characteristic feature of CRAM is a large extracellular domain with a roughly 66-fold acidic, cysteine-rich 12-amino-acid repeat. CRAM is conserved among different protozoan species, including Trypanosoma cruzi, and CRAM has structural similarities with eucaryotic cell surface receptors. The most striking homology of CRAM is to the human low-density-lipoprotein receptor. We propose that CRAM functions as a cell surface receptor of different trypanosome species.

scholarly journals Characterization of the R162W Kir7.1 mutation associated with snowflake vitreoretinopathy

2013 ◽  
Vol 304 (5) ◽  
pp. C440-C449 ◽  
Author(s):  
Wei Zhang ◽  
Xiaoming Zhang ◽  
Hui Wang ◽  
Anil K. Sharma ◽  
Albert O. Edwards ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Expression System ◽  
Dominant Negative ◽  
Dominant Negative Effect ◽  
Xenopus Laevis Oocyte ◽  
Cation Selectivity ◽  
Negative Effect ◽  
Dominant Disease ◽  
Vitreoretinal Degeneration ◽  
Inwardly Rectifying

KCNJ13 encodes Kir7.1, an inwardly rectifying K+ channel that is expressed in multiple ion-transporting epithelia. A mutation in KCNJ13 resulting in an arginine-to-tryptophan change at residue 162 (R162W) of Kir7.1 was associated with snowflake vitreoretinal degeneration, an inherited autosomal-dominant disease characterized by vitreous degeneration and mild retinal degeneration. We used the Xenopus laevis oocyte expression system to assess the functional properties of the R162W (mutant) Kir7.1 channel and determine how wild-type (WT) Kir7.1 is affected by the presence of the mutant subunit. Recordings obtained via the two-electrode voltage-clamp technique revealed that injection of oocytes with mutant Kir7.1 cRNA resulted in currents and cation selectivity that were indistinguishable from those in water-injected oocytes, suggesting that the mutant protein does not form functional channels in the plasma membrane. Coinjection of oocytes with equal amounts of mutant and WT Kir7.1 cRNAs resulted in inward K+ and Rb+ currents with amplitudes that were ∼17% of those in oocytes injected with WT Kir7.1 cRNA alone, demonstrating a dominant-negative effect of the mutant subunit. Similar to oocytes injected with WT Kir7.1 cRNA alone, coinjected oocytes exhibited inwardly rectifying Rb+ currents that were more than seven times larger than K+ currents, indicating that mutant subunits did not alter Kir7.1 channel selectivity. Immunostaining of Xenopus oocytes or Madin-Darby canine kidney cells expressing mutant or WT Kir7.1 demonstrated distribution of both proteins primarily in the plasma membrane. Our data suggest that the R162W mutation suppresses Kir7.1 channel activity, possibly by negatively impacting gating by membrane phosphadidylinositol 4,5-bisphosphate.

scholarly journals Cellular encoding of Cy dyes for single-molecule imaging

eLife ◽  
2016 ◽  
Vol 5 ◽  
Author(s):  
Lilia Leisle ◽  
Rahul Chadda ◽  
John D Lueck ◽  
Daniel T Infield ◽  
Jason D Galpin ◽  
...  
Keyword(s):  
Single Molecule ◽  
Expression System ◽  
Eukaryotic Expression ◽  
Cyanine Dye ◽  
Nonsense Suppression ◽  
Xenopus Laevis Oocyte ◽  
Cellular Environment ◽  
Oocyte Expression System ◽  
Improved Technique

A general method is described for the site-specific genetic encoding of cyanine dyes as non-canonical amino acids (Cy-ncAAs) into proteins. The approach relies on an improved technique for nonsense suppression with in vitro misacylated orthogonal tRNA. The data show that Cy-ncAAs (based on Cy3 and Cy5) are tolerated by the eukaryotic ribosome in cell-free and whole-cell environments and can be incorporated into soluble and membrane proteins. In the context of the Xenopus laevis oocyte expression system, this technique yields ion channels with encoded Cy-ncAAs that are trafficked to the plasma membrane where they display robust function and distinct fluorescent signals as detected by TIRF microscopy. This is the first demonstration of an encoded cyanine dye as a ncAA in a eukaryotic expression system and opens the door for the analysis of proteins with single-molecule resolution in a cellular environment.

scholarly journals Induction of GLUTAMINE DUMPER1 reveals a link between amino acid export, abscisic acid, and immune responses

2020 ◽  
Author(s):  
Shi Yu ◽  
Delasa Aghamirzaie ◽  
Kim Harich ◽  
Eva Collakova ◽  
Ruth Grene ◽  
...  
Keyword(s):  
Abscisic Acid ◽  
Amino Acid ◽  
Immune Responses ◽  
Transmembrane Domain ◽  
Induced Defense ◽  
Expression System ◽  
Amino Acid Content ◽  
Defense Responses ◽  
Developmental Needs ◽  
Amino Acid Homeostasis

ABSTRACTAmino acid homeostasis in plants is finely tuned to match developmental needs and response to adverse environments. Over-expression of the single-transmembrane domain protein GLUTAMINE DUMPER1 (GDU1) leads to increased amino acid export, reduced growth and constitutive induction of immune responses. We used an inducible gene expression system to tease apart the primary and secondary effects caused by GDU1, and demonstrated that the primary effect is increasing amino acid export, followed by increased amino acid content and abscisic acid (ABA) response, and a subsequent activation of defense responses. The GDU1-mediated hypersensitivity to ABA partially depended on the E3 ubiquitin ligase LOSS-OF-GDU1 2 (LOG2), a known GDU1 interactor. More importantly, the lysine catabolite pipecolic acid played a pivotal role in the GDU1-induced defense responses. This work unravels a novel relationship between amino acid transport, ABA and defense responses, potentially mediated by the GDU1-LOG2 complex, critical for understanding how plants respond to amino acid imbalance.ONE SENTENCE SUMMARYGenetically induced disturbance of amino acid homeostasis sequentially triggers responses to abiotic stresses and plant defenses to pathogens in Arabidopsis through undefined sensing mechanisms

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