scholarly journals Zip14 is a complex broad-scope metal-ion transporter whose functional properties support roles in the cellular uptake of zinc and nontransferrin-bound iron

2011 ◽  
Vol 301 (4) ◽  
pp. C862-C871 ◽  
Author(s):  
Jorge J. Pinilla-Tenas ◽  
Brian K. Sparkman ◽  
Ali Shawki ◽  
Anthony C. Illing ◽  
Colin J. Mitchell ◽  
...  
Keyword(s):  
Functional Properties ◽  
Cellular Uptake ◽  
Metal Ion ◽  
Transmembrane Protein ◽  
Expression System ◽  
Nitrilotriacetic Acid ◽  
Isolated Hepatocytes ◽  
Xenopus Laevis Oocyte ◽  
Ion Transporter ◽  
Broad Scope

Recent studies have shown that overexpression of the transmembrane protein Zrt- and Irt-like protein 14 (Zip14) stimulates the cellular uptake of zinc and nontransferrin-bound iron (NTBI). Here, we directly tested the hypothesis that Zip14 transports free zinc, iron, and other metal ions by using the Xenopus laevis oocyte heterologous expression system, and use of this approach also allowed us to characterize the functional properties of Zip14. Expression of mouse Zip14 in RNA-injected oocytes stimulated the uptake of 55Fe in the presence of l-ascorbate but not nitrilotriacetic acid, indicating that Zip14 is an iron transporter specific for ferrous ion (Fe2+) over ferric ion (Fe3+). Zip14-mediated 55Fe2+ uptake was saturable ( K0.5 ≈ 2 μM), temperature-dependent (apparent activation energy, Ea = 15 kcal/mol), pH-sensitive, Ca2+-dependent, and inhibited by Co2+, Mn2+, and Zn2+. HCO3− stimulated 55Fe2+ transport. These properties are in close agreement with those of NTBI uptake in the perfused rat liver and in isolated hepatocytes reported in the literature. Zip14 also mediated the uptake of 109Cd2+, 54Mn2+, and 65Zn2+ but not 64Cu (I or II). 65Zn2+ uptake also was saturable ( K0.5 ≈ 2 μM) but, notably, the metal-ion inhibition profile and Ca2+ dependence of Zn2+ transport differed from those of Fe2+ transport, and we propose a model to account for these observations. Our data reveal that Zip14 is a complex, broad-scope metal-ion transporter. Whereas zinc appears to be a preferred substrate under normal conditions, we found that Zip14 is capable of mediating cellular uptake of NTBI characteristic of iron-overload conditions.

Multiple epithelial Na+ channel domains participate in subunit assembly

2003 ◽  
Vol 285 (4) ◽  
pp. F600-F609 ◽  
Author(s):  
James B. Bruns ◽  
Baofeng Hu ◽  
Yoon J. Ahn ◽  
Shaohu Sheng ◽  
Rebecca P. Hughey ◽  
...  
Keyword(s):  
Transmembrane Protein ◽  
Expression System ◽  
Functional Expression ◽  
Full Length ◽  
Na Channel ◽  
Xenopus Laevis Oocyte ◽  
Subunit Interactions ◽  
Α Subunit ◽  
Hydrophobic Domain ◽  
Membrane Spanning

Epithelial sodium channels (ENaCs) are composed of three structurally related subunits that form a tetrameric channel. The Xenopus laevis oocyte expression system was used to identify regions within the ENaC α-subunit that confer a dominant negative phenotype on functional expression of αβγ-ENaC to define domains that have a role in subunit-subunit interactions. Coexpression of full-length mouse αβγ-ENaC with either 1) the α-subunit first membrane-spanning domain and short downstream hydrophobic domain (α-M1H1); 2) α-M1H1 and its downstream hydrophilic extracellular loop (α-M1H1-ECL); 3) the membrane-spanning domain of a control type 2 transmembrane protein (glutamyl transpeptidase; γ-GT) fused to the α-ECL (γ-GT-α-ECL); 4) the extracellular domain of a control type 1 transmembrane protein (Tac) fused to the α-subunit second membrane-spanning domain and short upstream hydrophobic domain (Tac-α-H2M2); or 5) the α-subunit cytoplasmic COOH terminus (α-Ct) significantly reduced amiloride-sensitive Na+ currents in X. laevis oocytes. Functional expression of Na+ channels was not inhibited when full-length αβγ-ENaC was coexpressed with either 1) the α-ECL lacking a signal-anchor sequence, 2) α-M1H1 and α-Ct expressed as a fusion protein, 3) full-length γ-GT, or 4) full-length Tac. Furthermore, the expression of ROMK channels was not inhibited when full-length ROMK was coexpressed with either α-M1H1-ECL or α-Ct. Full-length FLAG-tagged α-, β-, or γ-ENaC coimmunoprecipitated with myc-tagged α-M1H1-ECL, whereas wild-type γ-GT did not. These data suggest that multiple sites within the α-subunit participate in subunit-subunit interactions that are required for proper assembly of the heterooligomeric ENaC complex.

scholarly journals Functional properties of multiple isoforms of human divalent metal-ion transporter 1 (DMT1)

2007 ◽  
Vol 403 (1) ◽  
pp. 59-69 ◽  
Author(s):  
Bryan Mackenzie ◽  
Hitomi Takanaga ◽  
Nadia Hubert ◽  
Andreas Rolfs ◽  
Matthias A. Hediger
Keyword(s):  
Plasma Membrane ◽  
Functional Properties ◽  
Metal Ion ◽  
Cell Types ◽  
Divalent Metal ◽  
The Body ◽  
Ion Transporter ◽  
Voltage Dependent ◽  
Divalent Metal Ion ◽  
Responsive Element

DMT1 (divalent metal-ion transporter 1) is a widely expressed metal-ion transporter that is vital for intestinal iron absorption and iron utilization by most cell types throughout the body, including erythroid precursors. Mutations in DMT1 cause severe microcytic anaemia in animal models. Four DMT1 isoforms that differ in their N- and C-termini arise from mRNA transcripts that vary both at their 5′-ends (starting in exon 1A or exon 1B) and at their 3′-ends giving rise to mRNAs containing (+) or lacking (−) the 3′-IRE (iron-responsive element) and resulting in altered C-terminal coding sequences. To determine whether these variations result in functional differences between isoforms, we explored the functional properties of each isoform using the voltage clamp and radiotracer assays in cRNA-injected Xenopus oocytes. 1A/IRE(+)-DMT1 mediated Fe2+-evoked currents that were saturable (K0.5Fe≈1–2 μM), temperature-dependent (Q10≈2), H+-dependent (K0.5H≈1 μM) and voltage-dependent. 1A/IRE(+)-DMT1 exhibited the provisional substrate profile (ranked on currents) Cd2+, Co2+, Fe2+, Mn2+>Ni2+, V3+≫Pb2+. Zn2+ also evoked large currents; however, the zinc-evoked current was accounted for by H+ and Cl− conductances and was not associated with significant Zn2+ transport. 1B/IRE(+)-DMT1 exhibited the same substrate profile, Fe2+ affinity and dependence on the H+ electrochemical gradient. Each isoform mediated 55Fe2+ uptake and Fe2+-evoked currents at low extracellular pH. Whereas iron transport activity varied markedly between the four isoforms, the activity for each correlated with the density of anti-DMT1 immunostaining in the plasma membrane, and the turnover rate of the Fe2+ transport cycle did not differ between isoforms. Therefore all four isoforms of human DMT1 function as metal-ion transporters of equivalent efficiency. Our results reveal that the N- and C-terminal sequence variations among the DMT1 isoforms do not alter DMT1 functional properties. We therefore propose that these variations serve as tissue-specific signals or cues to direct DMT1 to the appropriate subcellular compartments (e.g. in erythroid cells) or the plasma membrane (e.g. in intestine).

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 Intestinal divalent metal‐ion transporter‐1 is critical for iron homeostasis but is not required for maintenance of Cu or Zn

2012 ◽  
Vol 26 (S1) ◽  
Author(s):  
Ali Shawki ◽  
Sarah R Anthony ◽  
Yasuhiro Nose ◽  
Tomasa Barrientos De Renshaw ◽  
Dennis J Thiele ◽  
...  
Keyword(s):  
Metal Ion ◽  
Iron Homeostasis ◽  
Divalent Metal ◽  
Ion Transporter ◽  
Divalent Metal Ion

scholarly journals Recombinant Diabody-Based Immunocapture Enzyme-Linked Immunosorbent Assay for Quantification of Rabies Virus Glycoprotein

2010 ◽  
Vol 17 (8) ◽  
pp. 1261-1268 ◽  
Author(s):  
Sridevi V. Nimmagadda ◽  
Shukra M. Aavula ◽  
Neelakantam Biradhar ◽  
Varaprasada Sankarasetty Rao ◽  
Rajalakshmi Shanmugham ◽  
...  
Keyword(s):  
Rabies Virus ◽  
Immunosorbent Assay ◽  
Human Monoclonal Antibody ◽  
Expression System ◽  
Nitrilotriacetic Acid ◽  
Human Rabies ◽  
Enzyme Linked Immunosorbent Assay ◽  
Rabies Virus Glycoprotein ◽  
Virus Glycoprotein ◽  
Overlap Extension

ABSTRACT The potency of rabies vaccines, determined using the NIH mouse protection test, can be directly correlated to the amount of rabies virus glycoprotein (RV GP) present in the vaccine. In an effort to develop a simple and sensitive enzyme-linked immunosorbent assay (ELISA) using recombinant diabody for quantification of RV GP, the variable heavy (VH) and light chain (VL) domains of an RV GP-specific human monoclonal antibody (MAb) secreted by a human × mouse heterohybridoma (human MAb R16E5) was amplified, linked using splicing by overlap extension PCR (SOE PCR), and expressed as a recombinant diabody (D06) in the pET28a bacterial expression system. The diabody D06 was purified by immobilized metal affinity chromatography on a nickel-nitrilotriacetic acid (NTA) agarose column and characterized. The purified diabody was used in combination with a well-characterized RV GP-specific mouse MAb, M5B4, to develop an immunocapture ELISA (IC-ELISA) for the quantification of RV GP in human rabies vaccine preparations. The maximum detection limit of the IC-ELISA using the M5B4-D06 combination was up to 31.25 ng/ml of RV GP. The specificity of the diabody was established by its nonreactivity toward other human viral antigens as determined by ELISA and toward RV GP as determined by immunoblot transfer assay and competitive ELISA with the parent human MAb R16E5 and MAb M5B4. The adjusted r 2 value obtained by the regression through the origin model was 0.902, and the equation for predicted potency values for M5B4-D06-based IC-ELISA and MAb M5B4 IC-ELISA were 0.5651x and 0.8044x, respectively, where x is the estimate of RV GP from the IC-ELISA in micrograms. Analysis of variance (ANOVA) results showed the estimates of the two methods differed significantly (P < 0.001), while the predicted potencies by the two tests did not differ significantly (P > 0.05). The IC-ELISA can be readily adapted to measure the RV GP content in purified antigen, and a vaccine can be formulated based on the estimated GP.

Contribution of monocarboxylate transporter 12 to blood supply of creatine on the sinusoidal membrane of the hepatocytes

2021 ◽  
Author(s):  
Ryuta Jomura ◽  
Yu Tanno ◽  
Shin-ichi Akanuma ◽  
Yoshiyuki Kubo ◽  
Masanori Tachikawa ◽  
...  
Keyword(s):  
Energy Homeostasis ◽  
Expression System ◽  
Circulatory System ◽  
Rat Hepatocytes ◽  
High Energy ◽  
The Body ◽  
Monocarboxylate Transporter ◽  
Xenopus Laevis Oocyte ◽  
Inflammatory Stress ◽  
Intracellular Energy

Creatine (Cr)/phosphocreatine has the ability to buffer the high-energy phosphate, thereby contributing to intracellular energy homeostasis. As Cr biosynthetic enzyme deficiency is reported to increase susceptibility to colitis under conditions of inflammatory stress, Cr is critical for maintaining intestinal homeostasis under inflammatory stress. Cr is mainly produced in the hepatocytes and then distributed to other organs of the body by the circulatory system. Since monocarboxylate transporter 9 (MCT9) and MCT12 have been reported to accept Cr as a substrate, these transporters are proposed as candidates for Cr efflux transporter in the liver. The aim of this study was to elucidate the transport mechanism on Cr supply from the hepatocytes. Immunohistochemical staining of the rat liver sections revealed that both MCT9 and MCT12 were localized on the sinusoidal membrane of the hepatocytes. In the transport studies using Xenopus laevis oocyte expression system, [14C]Cr efflux from MCT9- or MCT12-expressing oocytes was significantly greater than that from water-injected oocytes. [14C]Cr efflux from primary cultured hepatocytes was significantly decreased following MCT12 mRNA knockdown, whereas this efflux was not decreased after mRNA knockdown of MCT9. Based on the extent of MCT12 protein downregulation and Cr efflux after knockdown of MCT12 in primary cultured rat hepatocytes, the contribution ratio of MCT12 in Cr efflux was calculated as 76.4%. Our study suggests that MCT12 substantially contributes to the efflux of Cr at the sinusoidal membrane of the hepatocytes.

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