Mutation of a conserved proline residue in the beta-subunit ectodomain prevents Na(+)-K(+)-ATPase oligomerization

1993 ◽  
Vol 265 (4) ◽  
pp. C1169-C1174 ◽  
Author(s):  
K. Geering ◽  
P. Jaunin ◽  
F. Jaisser ◽  
A. M. Merillat ◽  
J. D. Horisberger ◽  
...  
Keyword(s):  
Sodium Pump ◽  
Critical Role ◽  
Pump Current ◽  
Beta Subunit ◽  
Sequence Motif ◽  
Wild Type ◽  
Ouabain Binding ◽  
Conserved Sequence ◽  
External Potassium ◽  
And Function

A highly conserved sequence motif (4 tyrosines and 1 proline: YYPYY) of the Na(+)-K(+)-adenosinetriphosphatase (ATPase) beta 1-subunit ectodomain has been mutagenized to study its possible role in alpha/beta-assembly and sodium pump function. Single as well as double tyrosine mutants (tyrosine to phenylalanine: Y to F) of Xenopus laevis beta 1-subunits are able to associate with alpha 1-subunits and form functional Na-K pumps at the plasma membrane that are indistinguishable from wild-type alpha 1, beta 1-Na-K pumps (as assessed by measurements of ouabain binding, 86Rb flux, Na-K pump current, and activation by external potassium). In contrast, a single proline mutation (proline to glycine: P244G) reduced by > 90% the proper assembly and function of Na(+)-K(+)-ATPase, despite a normal rate of synthesis and core glycosylation. Our data indicate that proline-244 plays a critical role in the proper folding of the beta-subunit and its ability to associate efficiently with the alpha 1-subunit in the endoplasmic reticulum.

Electric current generated by squid giant axon sodium pump: external K and internal ADP effects

1978 ◽  
Vol 235 (1) ◽  
pp. C63-C68 ◽  
Author(s):  
R. F. Abercrombie ◽  
P. de Weer
Keyword(s):  
Sodium Pump ◽  
Giant Axon ◽  
Pump Current ◽  
Membrane Resistance ◽  
Extracellular Potassium ◽  
Sodium Efflux ◽  
Steroid Sensitive ◽  
External Potassium ◽  
Loligo Pealei ◽  
External K

The operation of the sodium pump of giant axons of the squid, Loligo pealei, has been studied simultaneously in two independent ways: 1) by measuring sodium efflux with 22Na, and 2) by calculating the transmembrane current generated by the pump from measurements of membrane resistance and digitalis-sensitive membrane potential. In normal, untreated axons, the effect of increasing the external potassium concentration on both sodium efflux and pump current is similar, which suggests that Na:K pump stoichiometry remains relatively constant in the range of 0-20 mM external K. The data are compatible with a 3:2 Na:K ratio. In axons whose intracellular ADP level has been elevated by injection of L-arginine, a large, electrically silent, cardiotonic steroid-sensitive sodium efflux takes place in the absence of external potassium; this suggests that pump-mediated Na:Na exchange is 1:1 or electroneutral. Finally, elevation of external potassium levels causes the appearance, in high-ADP axons, of electrogenic pumping, with little effect on sodium efflux; hence, in contrast to what is seen in normal (low-ADP) axons, the charge translocated, per sodium ion extruded, increases sharply with increasing extracellular potassium levels.

scholarly journals Interactions between Brucella suis VirB8 and Its Homolog TraJ from the Plasmid pSB102 Underline the Dynamic Nature of Type IV Secretion Systems

2009 ◽  
Vol 191 (9) ◽  
pp. 2985-2992 ◽  
Author(s):  
Gisèle Bourg ◽  
Romain Sube ◽  
David O'Callaghan ◽  
Gilles Patey
Keyword(s):  
Critical Role ◽  
Type Iv Secretion ◽  
Dominant Negative Effect ◽  
Wild Type ◽  
Secretion Systems ◽  
Type Iv ◽  
Brucella Suis ◽  
Periplasmic Domain ◽  
Tm Domain ◽  
And Function

ABSTRACT The proteinVirB8 plays a critical role in the assembly and function of the Agrobacterium tumefaciens virB type IV secretion system (T4SS). The structure of the periplasmic domain of both A. tumefaciens and Brucella suis VirB8 has been determined, and site-directed mutagenesis has revealed amino acids involved in the dimerization of VirB8 and interactions with VirB4 and VirB10. We have shown previously that TraJ, the VirB8 homologue from pSB102, and the chimeric protein TraJB8, encompassing the cytoplasmic and transmembrane (TM) domains of TraJ and the periplasmic domain of VirB8, were unable to complement a B. suis mutant containing an in-frame deletion of the virB8 gene. This suggested that the presence of the TraJ cytoplasmic and TM domains could block VirB8 dimerization or assembly in the inner membrane. By bacterial two-hybrid analysis, we found that VirB8, TraJ, and the chimeras can all interact to form both homo- and heterodimers. However, the presence of the TM domain of TraJ resulted in much stronger interactions in both the homo- and heterodimers. We expressed the wild-type and chimeric proteins in wild-type B. suis. The presence of proteins carrying the TM domain of TraJ had a dominant negative effect, leading to complete loss of virulence. This suggests that the T4SS is a dynamic structure and that strong interactions block the spatial flexibility required for correct assembly and function.

Dilated cardiomyopathy in Erb-b4-deficient ventricular muscle

2005 ◽  
Vol 289 (3) ◽  
pp. H1153-H1160 ◽  
Author(s):  
Hernán García-Rivello ◽  
Julián Taranda ◽  
Matilde Said ◽  
Patricia Cabeza-Meckert ◽  
Martin Vila-Petroff ◽  
...  
Keyword(s):  
Dilated Cardiomyopathy ◽  
Receptor Tyrosine Kinase ◽  
Cardiac Development ◽  
Critical Role ◽  
Premature Death ◽  
Ventricular Muscle ◽  
Wild Type ◽  
Eccentric Hypertrophy ◽  
And Function ◽  
Intercellular Communications

The neuregulin receptor tyrosine kinase Erb-b4, initially linked to early cardiac development, is shown here to play a critical role in adult cardiac function. In wild-type mice, Erb-b4 protein localized to Z lines and to intercalated disks, suggesting a role in subcellular and intercellular communications of cardiomyocytes. Conditional inactivation of erb-b4 in ventricular muscle cells led to a severe dilated cardiomyopathy, characterized by thinned ventricular walls with eccentric hypertrophy, reduced contractility, and delayed conduction. This cardiac dysfunction may account for premature death in adult erb-b4-knockout mice. This study establishes a critical role for Erb-b4 in the maintenance of normal postnatal cardiac structure and function.

scholarly journals Acute pH-dependent Regulation of AE2-mediated Anion Exchange Involves Discrete Local Surfaces of the NH2-terminal Cytoplasmic Domain

2004 ◽  
Vol 279 (50) ◽  
pp. 52664-52676 ◽  
Author(s):  
Andrew K. Stewart ◽  
Nicky Kerr ◽  
Marina N. Chernova ◽  
Seth L. Alper ◽  
Richard D. Vaughan-Jones
Keyword(s):  
Amino Acid ◽  
Critical Role ◽  
Surface Region ◽  
Cytoplasmic Domain ◽  
Ph Sensitivity ◽  
Dimensional Structure ◽  
Disulfonic Acid ◽  
Wild Type ◽  
Conserved Sequence ◽  
Ph Dependent

We have previously defined in the NH2-terminal cytoplasmic domain of the mouse AE2/SLC4A2 anion exchanger a critical role for the highly conserved amino acids (aa) 336–347 in determining wild-type pH sensitivity of anion transport. We have now engineered hexa-Ala ((A)6) and individual amino acid substitutions to investigate the importance to pH-dependent regulation of AE2 activity of the larger surrounding region of aa 312–578. 4,4′-Diisothiocyanostilbene-2,2′-disulfonic acid (DIDS)-sensitive36Cl-efflux from AE2-expressingXenopusoocytes was monitored during changes in pHior pHoin HEPES-buffered and in 5%\batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{CO}_{2}{/}\mathrm{HCO}_{3}^{-}\) \end{document}-buffered conditions. Wild-type AE2-mediated36Cl-efflux was profoundly inhibited at low pHo, with a pHo(50)value = 6.75 ± 0.05 and was stimulated up to 10-fold by intracellular alkalinization. Individual mutation of several amino acid residues at non-contiguous sites preceding or following the conserved sequence aa 336–347 attenuated pHiand/or pHosensitivity of36Cl-efflux. The largest attenuation of pH sensitivity occurred with the AE2 mutant (A)6357–362. This effect was phenocopied by AE2 H360E, suggesting a crucial role for His360. Homology modeling of the three-dimensional structure of the AE2 NH2-terminal cytoplasmic domain (based on the structure of the corresponding region of human AE1) predicts that those residues shown by mutagenesis to be functionally important define at least one localized surface region necessary for regulation of AE2 activity by pH.

scholarly journals Regulation and Function of Syk Tyrosine Kinase in Mast Cell Signaling and Beyond

2011 ◽  
Vol 2011 ◽  
pp. 1-9 ◽  
Author(s):  
Rodrigo Orlandini de Castro
Keyword(s):  
Mast Cell ◽  
Mast Cells ◽  
Tyrosine Kinase ◽  
Tyrosine Phosphorylation ◽  
Conformational Changes ◽  
Protein Tyrosine Kinase ◽  
Critical Role ◽  
Wild Type ◽  
And Function

The protein tyrosine kinase Syk plays a critical role in FcεRI signaling in mast cells. Binding of Syk to phosphorylated immunoreceptor tyrosine-based activation motifs (p-ITAM) of the receptor subunits results in conformational changes and tyrosine phosphorylation at multiple sites that leads to activation of Syk. The phosphorylated tyrosines throughout the molecule play an important role in the regulation of Syk-mediated signaling. Reconstitution of receptor-mediated signaling in Syk-/- cells by wild-type Syk or mutants which have substitution of these tyrosines with phenylalanine together with in vitro assays has been useful strategies to understand the regulation and function of Syk.

Evolution and structural diversification of hyperpolarization-activated cyclic nucleotide-gated channel genes

2007 ◽  
Vol 29 (3) ◽  
pp. 231-245 ◽  
Author(s):  
Heather A. Jackson ◽  
Christian R. Marshall ◽  
Eric A. Accili
Keyword(s):  
Cyclic Nucleotide ◽  
Phylogenetic Analyses ◽  
Critical Role ◽  
Hcn Channels ◽  
Ancestral Gene ◽  
Conserved Sequence ◽  
Gated Channel ◽  
Duplication Events ◽  
The Individual ◽  
And Function

Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels are members of the voltage-gated channel superfamily and play a critical role in cellular pace-making. Overall sequence conservation is high throughout the family, and channel functions are similar but not identical. Phylogenetic analyses are imperative to understand how these genes have evolved and to make informed comparisons of HCN structure and function. These have been previously limited, however, by the small number of available sequences, from a minimal number of species unevenly distributed over evolutionary time. We have now identified and annotated 31 novel genes from invertebrates, urochordates, fish, amphibians, birds, and mammals. With increased sequence numbers and a broader species representation, a more precise sequence comparison was performed and an evolutionary history for these genes was constructed. Our data confirm the existence of at least four vertebrate paralogs and suggest that these arose via three duplication and diversification events from a single ancestral gene. Additional lineage-specific duplications appear to have occurred in urochordate and fish genomes. Based on exon boundary conservation and phylogenetic analyses, we hypothesize that mammalian gene structure was established, and duplication events occurred, after the divergence of urochordates and before the divergence of fish from the tetrapod lineage. In addition, we identified highly conserved sequence regions that are likely important for general HCN functions, as well as regions with differences conserved among each of the individual paralogs. The latter may underlie more subtle isoform-specific properties that are otherwise masked by the high identity among mammalian orthologs and/or inaccurate alignments between paralogs.

Flt3-Ligand Plays a Critical Role in Development and Function of CD8+/TCR− Facilitating Cells in Bone Marrow,

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 4003-4003
Author(s):  
Yiming Huang ◽  
Thomas Miller ◽  
Hong Xu ◽  
Yujie Wen ◽  
Suzanne T Ildstad
Keyword(s):  
Bone Marrow ◽  
Critical Role ◽  
Flt3 Ligand ◽  
Wild Type ◽  
Graft Versus Host ◽  
Equity Ownership ◽  
Total Body ◽  
And Function

Abstract Abstract 4003 Graft facilitating cells (FC) are a CD8+/TCR− bone marrow subpopulation that enhance engraftment of purified hematopoietic cells (HSC) in allogeneic mouse recipients without causing graft-versus-host disease. They also enhance engraftment of suboptimal numbers of syngeneic HSC. FC induce antigen-specific CD4+/CD25+/FoxP3+ regulatory T cells in vivo. The major subpopulation in FC is resembles plasmacytoid precursor dendritic cells (p-preDC) both phenotypically and functionally. Treatment of mice with Flt3 ligand (FL) results in a significant increase in FC in peripheral blood (PB) and FL-expanded-PB FC enhanced HSC engraftment. In this study, we evaluated the role of FL in FC development using FL-KO mice. We first compared FC from FL-KO B6 mice with FC from B6 mice to evaluate the FC total cellular composition. The number of FC was significantly decreased in FL-KO mice compared to wild type controls (P = 0.0003). The number of p-preDC FC was also significantly decreased (P = 0.0001), suggesting that FL is important in the development of p-preDC FC. Next, we tested whether FL-KO FC facilitate engraftment of HSC in allogeneic recipients. FC were sorted from FL-KO B6 mice and HSC (C-Kit+/Sca-1+/Lin−) were sorted from B6 mice. 10,000 B6 HSC plus 30,000 FL-KO FC were transplanted into NOD recipients conditioned with 950 cGy of total body irradiation. Controls received 10,000 B6 HSC with or without 30,000 B6 FC. Only 36% (5 of 14) NOD recipients of B6 HSC alone engrafted and two mice survived up to 160 days (Figure). Sixty-three percent (5 of 8) of recipients transplanted with B6 HSC + FL-KO B6 FC engrafted and only one mouse survived up to 160 days. Seventy-five percent (9 of 12) recipients of B6 HSC + B6 FC engrafted and seven of the mice survived more than 160 days. The level of donor chimerism in recipients of B6 HSC + B6 FC (57% ± 10%) was significantly higher than recipients of B6 HSC + FL-KO B6 FC (14% ± 3%; P = 0.003) or B6 HSC alone (22% ± 6%; P = 0.005). These data demonstrate that FL-KO FC fail to facilitate durable allogeneic HSC engraftment, suggesting that flt3-ligand plays a critical role in development of functional FC. Disclosures: Ildstad: Regenerex, LLC: Equity Ownership.

scholarly journals CD11b regulates obesity-induced insulin resistance via limiting alternative activation and proliferation of adipose tissue macrophages

2015 ◽  
Vol 112 (52) ◽  
pp. E7239-E7248 ◽  
Author(s):  
Chunxing Zheng ◽  
Qian Yang ◽  
Chunliang Xu ◽  
Peishun Shou ◽  
Jianchang Cao ◽  
...  
Keyword(s):  
Insulin Resistance ◽  
Adipose Tissue ◽  
High Fat Diet ◽  
Critical Role ◽  
Alternative Activation ◽  
Wild Type ◽  
Adipose Tissue Macrophages ◽  
Monocytes And Macrophages ◽  
And Function ◽  
Deficient Mice

Obesity-associated inflammation is accompanied by the accumulation of adipose tissue macrophages (ATMs), which is believed to predispose obese individuals to insulin resistance. CD11b (integrin αM) is highly expressed on monocytes and macrophages and is critical for their migration and function. We found here that high-fat diet–induced insulin resistance was significantly reduced in CD11b-deficient mice. Interestingly, the recruitment of monocytes to adipose tissue is impaired when CD11b is deficient, although the cellularity of ATMs in CD11b-deficient mice is higher than that in wild-type mice. We further found that the increase in ATMs is caused mainly by their vigorous proliferation in the absence of CD11b. Moreover, the proliferation and alternative activation of ATMs are regulated by the IL-4/STAT6 axis, which is inhibited by CD11b through the activity of phosphatase SHP-1. Thus, CD11b plays a critical role in obesity-induced insulin resistance by limiting the proliferation and alternative activation of ATMs.

scholarly journals Structure and Function of the Sodium Pump. The Role of the .BETA. subunit.

MEMBRANE ◽  
1995 ◽  
Vol 20 (2) ◽  
pp. 115-125
Author(s):  
Susumu Ueno ◽  
Futoshi Izumi ◽  
Masaru Kawamura
Keyword(s):  
Sodium Pump ◽  
Structure And Function ◽  
Beta Subunit ◽  
And Function

scholarly journals Critical role of the first transmembrane domain of Cx26 in regulating oligomerization and function

2012 ◽  
Vol 23 (17) ◽  
pp. 3299-3311 ◽  
Author(s):  
Oscar Jara ◽  
Rodrigo Acuña ◽  
Isaac E. García ◽  
Jaime Maripillán ◽  
Vania Figueroa ◽  
...  
Keyword(s):  
Gap Junction ◽  
Transmembrane Domain ◽  
Critical Role ◽  
Full Length ◽  
Intermediate Step ◽  
Wild Type ◽  
Gap Junction Channels ◽  
Junction Protein ◽  
And Function ◽  
Pore Domain

To identify motifs involved in oligomerization of the gap junction protein Cx26, we studied individual transmembrane (TM) domains and the full-length protein. Using the TOXCAT assay for interactions of isolated TM α-helices, we found that TM1, a Cx26 pore domain, had a strong propensity to homodimerize. We identified amino acids Val-37–Ala-40 (VVAA) as the TM1 motif required for homodimerization. Two deafness-associated Cx26 mutations localized in this region, Cx26V37I and Cx26A40G, differentially affected dimerization. TM1-V37I dimerized only weakly, whereas TM1-A40G did not dimerize. When the full-length mutants were expressed in HeLa cells, both Cx26V37I and Cx26A40G formed oligomers less efficiently than wild-type Cx26. A Cx26 cysteine substitution mutant, Cx26V37C formed dithiothreitol-sensitive dimers. Substitution mutants of Val-37 formed intercellular channels with reduced function, while mutants of Ala-40 did not form functional gap junction channels. Unlike wild-type Cx26, neither Cx26V37I nor Cx26A40G formed functional hemichannels in low extracellular calcium. Thus the VVAA motif of Cx26 is critical for TM1 dimerization, hexamer formation, and channel function. The differential effects of VVAA mutants on hemichannels and gap junction channels imply that inter-TM interactions can differ in unapposed and docked hemichannels. Moreover, Cx26 oligomerization appears dependent on transient TM1 dimerization as an intermediate step.

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