scholarly journals Dominant-Negative Regulation of Cell Surface Expression by a Pentapeptide Motif at the Extreme COOH Terminus of an Slo1 Calcium-Activated Potassium Channel Splice Variant

2010 ◽  
Vol 77 (4) ◽  
pp. 497-507 ◽  
Author(s):  
Yu-Hsin Chiu ◽  
Claudia Alvarez-Baron ◽  
Eun Young Kim ◽  
Stuart E. Dryer
Keyword(s):  
Cell Surface ◽  
Potassium Channel ◽  
Splice Variant ◽  
Surface Expression ◽  
Negative Regulation ◽  
Dominant Negative ◽  
Cell Surface Expression ◽  
Calcium Activated Potassium Channel

Abstract 1070: An Unconventional Vesicular Transport Pathway Regulates the Cell Surface Expression of hERG K + Channels

Circulation ◽  
2007 ◽  
Vol 116 (suppl_16) ◽  
Author(s):  
Heather A Underkofler ◽  
Sadguna Balijepalli ◽  
Brooke M Moungey ◽  
Jessica K Slind ◽  
Jabe M Best ◽  
...  
Keyword(s):  
Cell Surface ◽  
Surface Membrane ◽  
Vesicular Transport ◽  
Surface Expression ◽  
Small Gtpases ◽  
Dominant Negative ◽  
Cell Surface Expression ◽  
Missense Mutations ◽  
Transport Pathway ◽  
Er Export

Approximately 35– 45% of patients that are genotype positive for congenital Long QT Syndrome (LQT) have mutations in the human Ether-a-go-go Related Gene ( hERG ). The purpose of this study was to elucidate the mechanisms that regulate ER export and cell surface expression of hERG channel protein, because these steps are impaired for ~90% of LQT-linked hERG missense mutations. The small GTPases Sar1 and Arf1 regulate the conventional vesicular transport (trafficking) for the ER export of proteins to the Golgi apparatus (Golgi). We generated dominant negative (DN) mutations for Sar1 and Arf1, and co-expressed these DN GTPases with hERG in HEK 293 cells. The trafficking of hERG through the Golgi can be visualized biochemically using Western blot analysis, because additional glycosylation of hERG in the Golgi (Golgi processing) increases the MW of hERG protein from 135kDa to 155kDa. Co-expression of hERG and DN-Sar1 inhibited Golgi processing, decreased hERG current (I hERG ) by 85% compared to control (n≥8 cells per group, p<0.05), and decreased the staining of hERG protein at the cell surface, while co-expression of hERG and DN-Arf1 showed no significant effect on Golgi processing or I hERG . This lack of an effect by DN-Arf1 was selective for hERG as it efficiently blocked the transport of previously reported proteins. Rab11 GTPases regulate the trafficking of proteins from endosomal compartments to the cell surface membrane and/or the Golgi. Rab11a is ubiquitously expressed, whereas Rab11b is expressed primarily in brain and heart. Co-expression of DN-Rab11a did not alter Golgi processing of hERG but reduced I hERG by 51% compared to control (n≥8 cells per group, p<0.05), whereas co-expression of DN-Rab11b inhibited Golgi processing of hERG and reduced I hERG by 79% compared to control (n=8 cells per group, p<0.05). Thus, Rab11a appears to regulate the trafficking of hERG to the cell surface after processing in the Golgi, whereas Rab11b regulates the trafficking of hERG prior to processing in the Golgi. These data suggest that hERG does not traffic via the conventional pathway from the ER to the Golgi, but rather in an unconventional pathway from the ER to endosomal compartments prior to Rab11b-mediated transport to the Golgi and subsequent delivery to the cell membrane.

Glycosylation and Cell Surface Expression of Kv1.2 Potassium Channel are Regulated by Determinants in the Pore Region

2006 ◽  
Vol 31 (5) ◽  
pp. 589-596 ◽  
Author(s):  
Tetsuhiro Fujita ◽  
Iku Utsunomiya ◽  
Jin Ren ◽  
Yousuke Matsushita ◽  
Miwa Kawai ◽  
...  
Keyword(s):  
Cell Surface ◽  
Potassium Channel ◽  
Surface Expression ◽  
Cell Surface Expression ◽  
Pore Region

Identification of amino acids in the pore region of Kv1.2 potassium channel that regulate its glycosylation and cell surface expression

2010 ◽  
Vol 112 (4) ◽  
pp. 913-923 ◽  
Author(s):  
Iku Utsunomiya ◽  
Shinya Tanabe ◽  
Tomonori Terashi ◽  
Souichi Ikeno ◽  
Tadashi Miyatake ◽  
...  
Keyword(s):  
Amino Acids ◽  
Cell Surface ◽  
Potassium Channel ◽  
Surface Expression ◽  
Cell Surface Expression ◽  
Pore Region

COMMD1 downregulates the epithelial sodium channel through Nedd4–2

2010 ◽  
Vol 298 (6) ◽  
pp. F1445-F1456 ◽  
Author(s):  
Ying Ke ◽  
A. Grant Butt ◽  
Marianne Swart ◽  
Yong Feng Liu ◽  
Fiona J. McDonald
Keyword(s):  
Epithelial Cells ◽  
Cell Surface ◽  
Sodium Channel ◽  
Rat Kidney ◽  
Copper Metabolism ◽  
Epithelial Sodium Channel ◽  
Surface Expression ◽  
Dominant Negative ◽  
Cell Surface Expression ◽  
Xenopus Laevis Oocytes

The epithelial sodium channel (ENaC) is important for the long-term control of Na+ homeostasis and blood pressure. Our previous studies demonstrated that Copper Metabolism Murr1 Domain-containing protein 1 (COMMD1; previously known as Murr1), a protein involved in copper metabolism, inhibited amiloride-sensitive current in Xenopus laevis oocytes expressing ENaC ( J Biol Chem 279: 5429, 2004). In this study, we report that COMMD1 inhibits amiloride-sensitive current in mammalian epithelial cells expressing ENaC, that the COMM domain of COMMD1 is sufficient for this effect, and that knockdown of COMMD1 increases amiloride-sensitive current. COMMD1 is coexpressed with ENaC in rat kidney medulla cells. COMMD1 increased ubiquitin modification of ENaC and decreased its cell surface expression. COMMD1 abolished insulin-stimulated amiloride-sensitive current and attenuated the stimulation of current by activated serum and glucocorticoid-regulated kinase (SGK1). COMMD1 was found to interact with both SGK1 and Akt1/protein kinase B, and knockdown of COMMD1 enhanced the stimulatory effect of both SGK1 and Akt1 on amiloride-sensitive current. COMMD1's effects were reduced in the presence of ENaC proteins containing PY motif mutations, abolished in the presence of a dominant negative form of Nedd4–2, and knockdown of COMMD1 reduced the inhibitory effect of Nedd4–2 on ENaC, but did not enhance current when Nedd4–2 was knocked down. These data suggest that COMMD1 modulates Na+ transport in epithelial cells through regulation of ENaC cell surface expression and this effect is likely mediated via Nedd4–2.

scholarly journals Roles of the N- and C-termini of GLUT4 in endocytosis

2002 ◽  
Vol 115 (1) ◽  
pp. 131-140 ◽  
Author(s):  
Hadi Al-Hasani ◽  
Raghu K. Kunamneni ◽  
Kevin Dawson ◽  
Cynthia S. Hinck ◽  
Dirk Müller-Wieland ◽  
...  
Keyword(s):  
Cell Surface ◽  
Glucose Transporter ◽  
Surface Expression ◽  
Dominant Negative ◽  
Cell Surface Expression ◽  
Target Cells ◽  
Dileucine Motif ◽  
Intracellular Compartments ◽  
Targeting Signal

In insulin target cells, the predominantly expressed glucose transporter isoform GLUT4 recycles between distinct intracellular compartments and the plasma membrane. To characterize putative targeting signals within GLUT4 in a physiologically relevant cell type, we have analyzed the trafficking of hemagglutinin (HA)-epitope-tagged GLUT4 mutants in transiently transfected primary rat adipose cells. Mutation of the C-terminal dileucine motif (LL489/90) did not affect the cell-surface expression of HA-GLUT4. However, mutation of the N-terminal phenylalanine-based targeting sequence (F5) resulted in substantial increases, whereas deletion of 37 or 28 of the 44 C-terminal residues led to substantial decreases in cell-surface HA-GLUT4 in both the basal and insulin-stimulated states. Studies with wortmannin and coexpression of a dominant-negative dynamin GTPase mutant indicate that these effects appear to be primarily due to decreases and increases, respectively, in the rate of endocytosis. Yeast two-hybrid analyses revealed that the N-terminal phenylalanine-based targeting signal in GLUT4 constitutes a binding site for medium chain adaptins μ1, μ2, and μ3A, implicating a role of this motif in the targeting of GLUT4 to clathrin-coated vesicles.

scholarly journals RasGRP1 stimulation enhances ubiquitination and endocytosis of the sodium-chloride cotransporter

2010 ◽  
Vol 299 (2) ◽  
pp. F300-F309 ◽  
Author(s):  
Benjamin Ko ◽  
Erik-Jan Kamsteeg ◽  
Leslie L. Cooke ◽  
Lauren N. Moddes ◽  
Peter M. T. Deen ◽  
...  
Keyword(s):  
Sodium Chloride ◽  
Cell Surface ◽  
Phorbol Ester ◽  
Phorbol Esters ◽  
Surface Expression ◽  
Dominant Negative ◽  
Cell Surface Expression ◽  
Activity Regulation ◽  
Sodium Chloride Cotransporter ◽  
Radiotracer Uptake

The sodium-chloride cotransporter (NCC) is the principal salt-absorptive pathway in the distal convoluted tubule. Recently, we described a novel pathway of NCC regulation in which phorbol esters (PE) stimulate Ras guanyl-releasing protein 1 (RasGRP1), triggering a cascade ultimately activating ERK1/2 MAPK and decreasing NCC cell surface expression (Ko B, Joshi LM, Cooke LL, Vazquez N, Musch MW, Hebert SC, Gamba G, Hoover RS. Proc Natl Acad Sci USA 104: 20120–20125, 2007). Little is known about the mechanisms which underlie these effects on NCC activity. Regulation of NCC via changes in NCC surface expression has been reported, but endocytosis of NCC has not been demonstrated. In this study, utilizing biotinylation, internalization assays, and a dynamin dominant-negative construct, we demonstrate that the regulation of NCC by PE occurs via an enhancement in internalization of NCC and is dynamin dependent. In addition, immunoprecipitation of NCC and subsequent immunoblotting for ubiquitin showed increased ubiquitination of NCC with phorbol ester treatment. MEK1/2 inhibitors and gene silencing of RasGRP1 indicated that this effect was dependent on RasGRP1 and ERK1/2 activation. Inhibition of ubiquitination prevents any PE-mediated decrease in NCC surface expression as measured by biotinylation or NCC activity as measured by radiotracer uptake. These findings confirmed that the PE effect on NCC is mediated by endocytosis of NCC. Furthermore, ubiquitination of NCC is essential for this process and this ubiquitination is dependent upon RasGRP1-mediated ERK1/2 activation.

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