Glu496Ala polymorphism of human P2X7receptor does not affect its electrophysiological phenotype

2003 ◽  
Vol 284 (3) ◽  
pp. C749-C756 ◽  
Author(s):  
Wolfgang Boldt ◽  
Manuela Klapperstück ◽  
Cora Büttner ◽  
Sven Sadtler ◽  
Günther Schmalzing ◽  
...  
Keyword(s):  
Amino Acid ◽  
Critical Role ◽  
Amino Acid Position ◽  
Functional Expression ◽  
Hek293 Cells ◽  
Kinetic Properties ◽  
Loss Of Function ◽  
Ion Channel Properties ◽  
Gated Channel ◽  
Channel Properties

A glutamate to alanine exchange at amino acid position 496 of the human P2X7 receptor was recently shown to be associated with a loss of function in human B lymphocytes in terms of ATP-induced ethidium+ uptake, Ba2+ influx, and induction of apoptosis (Gu BJ, Zhang WY, Worthington RA, Sluyter R, Dao-Ung P, Petrou S, Barden JA, and Wiley JS. J Biol Chem 276: 11135–11142, 2001). Here we analyzed the effect of the Glu496 to Ala exchange on the channel properties of the human P2X7 receptor expressed in Xenopus oocytes with the two-microelectrode voltage-clamp technique. The amplitudes of ATP-induced whole cell currents characteristic of functional expression, kinetic properties including ATP concentration dependence, and permeation behavior were not altered by this amino acid exchange. Also in HEK293 cells, the Ala496 mutant mediated typical P2X7 receptor-dependent currents like the parent Glu496 hP2X7 receptor. Because the function of the P2X7 receptor as an ATP-gated channel for small cations including Ba2+ remained unaffected by this mutation, we conclude that Glu496 plays a critical role in pore formation but does not determine the ion channel properties of the human P2X7 receptor.

scholarly journals Critical role of amino acid position 343 of surfactant protein-D in the selective binding of glycolipids from Mycobacterium tuberculosis

Glycobiology ◽  
2009 ◽  
Vol 19 (12) ◽  
pp. 1473-1484 ◽  
Author(s):  
Tracy K Carlson ◽  
Jordi B Torrelles ◽  
Kelly Smith ◽  
Tim Horlacher ◽  
Riccardo Castelli ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Amino Acid ◽  
Critical Role ◽  
Amino Acid Position ◽  
Surfactant Protein ◽  
Surfactant Protein D ◽  
Selective Binding

Abstract 376: Nedd4 Regulated Bk Channels in Diabetes Mellitus

2017 ◽  
Vol 37 (suppl_1) ◽  
Author(s):  
DAI-MIN ZHANG ◽  
Shao-liang Chen ◽  
Yanrong Zhu ◽  
Peng Ye
Keyword(s):  
Diabetes Mellitus ◽  
Current Density ◽  
Knockout Mice ◽  
Vascular Function ◽  
Critical Role ◽  
Vascular Diseases ◽  
Bk Channels ◽  
Bk Channel ◽  
Hek293 Cells ◽  
Kinetic Properties

Big conductance calcium activated potassium(BK) channel plays a critical role in pathophysiological regulation of vascular function. Recent studies indicated that the expression reduction of BK channels in high glucose condition exacerbated vessel dilation, and led to coronary artery diseases, while BK channel expression was reserved in A-kinase anchoring protein(AKAP) knockout mice at same condition. Here, We are to investigate heterologous co-expression of Nedd4 ligase, ubiquitin protein ligase, and KCa1.1 in HEK293 cells. The result shown that co-expression reduced BK current density without modulation of kinetic properties as measured by path clamp techniques. Modulation of current density was dependent on ligase activity and was lost in AKAP knockout mice with diabetes mellitus. Taken together, our data disclose a novel mechanism of KCa1.1 channel regulation that NEDD4 decreased BK channels expression in diabetes mellitus depending on AKAP signal complexity. These findings provide a new insight into potential therapeutic target in vascular diseases, especially in diabetes mellitus.This work was supported by the National Natural Science Foundation of China(Grant No. 8137034)

scholarly journals Recruitment of folliculin to lysosomes supports the amino acid–dependent activation of Rag GTPases

2013 ◽  
Vol 202 (7) ◽  
pp. 1107-1122 ◽  
Author(s):  
Constance S. Petit ◽  
Agnes Roczniak-Ferguson ◽  
Shawn M. Ferguson
Keyword(s):  
Amino Acid ◽  
Critical Role ◽  
Direct Interaction ◽  
Loss Of Function ◽  
Interacting Protein ◽  
Rag Gtpases ◽  
New Findings ◽  
Pulmonary Cysts ◽  
Amino Acid Depletion ◽  
A Site

Birt-Hogg-Dubé syndrome, a human disease characterized by fibrofolliculomas (hair follicle tumors) as well as a strong predisposition toward the development of pneumothorax, pulmonary cysts, and renal carcinoma, arises from loss-of-function mutations in the folliculin (FLCN) gene. In this study, we show that FLCN regulates lysosome function by promoting the mTORC1-dependent phosphorylation and cytoplasmic sequestration of transcription factor EB (TFEB). Our results indicate that FLCN is specifically required for the amino acid–stimulated recruitment of mTORC1 to lysosomes by Rag GTPases. We further demonstrated that FLCN itself was selectively recruited to the surface of lysosomes after amino acid depletion and directly bound to RagA via its GTPase domain. FLCN-interacting protein 1 (FNIP1) promotes both the lysosome recruitment and Rag interactions of FLCN. These new findings define the lysosome as a site of action for FLCN and indicate a critical role for FLCN in the amino acid–dependent activation of mTOR via its direct interaction with the RagA/B GTPases.

scholarly journals Membrane chaperone Shr3 assists in folding amino acid permeases preventing precocious ERAD

2007 ◽  
Vol 176 (5) ◽  
pp. 617-628 ◽  
Author(s):  
Jhansi Kota ◽  
C. Fredrik Gilstring ◽  
Per O. Ljungdahl
Keyword(s):  
Amino Acid ◽  
Critical Role ◽  
Amino Acid Uptake ◽  
Functional Expression ◽  
Coupled Processes ◽  
Acid Uptake ◽  
Transmembrane Segments ◽  
Large Molecular Weight ◽  
Tightly Coupled ◽  
Amino Acid Permeases

The yeast endoplasmic reticulum (ER) membrane-localized chaperone Shr3 plays a critical role in enabling amino acid permeases (AAPs) to fold and attain proper structures required for functional expression at the plasma membrane. In the absence of Shr3, AAPs specifically accumulate in the ER, where despite the correct insertion of their 12 transmembrane segments (TMSs), they aggregate forming large molecular weight complexes. We show that Shr3 prevents aggregation and facilitates the functional assembly of independently coexpressed N- and C-terminal fragments of the general AAP Gap1. Shr3 interacts with and maintains the first five TMSs in a conformation that can posttranslationally assemble with the remaining seven TMSs. We also show that Doa10- and Hrd1-dependent ER-associated degradation (ERAD) pathways redundantly degrade AAP aggregates. In combination, doa10Δ hrd1Δ mutations stabilize AAP aggregates and partially suppress amino acid uptake defects of shr3 mutants. Consequently, in cells with impaired ERAD, AAPs are able to attain functional conformations independent of Shr3. These findings illustrate that folding and degradation are tightly coupled processes during membrane protein biogenesis.

Inactivation of a Frameshift TSH Receptor Variant Val711Phefs*18 is Due to Acquisition of a Hydrophobic Degron

2020 ◽  
Vol 106 (1) ◽  
pp. e265-e272
Author(s):  
Chiho Sugisawa ◽  
Makoto Ono ◽  
Kenichi Kashimada ◽  
Tomonobu Hasegawa ◽  
Satoshi Narumi
Keyword(s):  
Amino Acid ◽  
Congenital Hypothyroidism ◽  
Fluorescent Protein ◽  
Transmembrane Domain ◽  
Tsh Receptor ◽  
Hek293 Cells ◽  
Luciferase Reporter ◽  
Loss Of Function ◽  
Tail Region

Abstract Context Inactivating variants of thyrotropin (thyroid-stimulating hormone; TSH) receptor (TSHR) cause congenital hypothyroidism. More than 60 such variants have been reported so far, most of which were located in the extracellular or transmembrane domain. Objective We report the identification and characterization of a frameshift TSHR variant in the intracytoplasmic C-tail region. Methods Sequencing of TSHR was performed in a patient with congenital hypothyroidism. The functionality of the identified variants was assessed by expressing TSHR in HEK293 cells and measuring TSH-dependent activation of the cAMP–response element-luciferase reporter. A series of systematic mutagenesis experiments were performed to characterize the frameshifted amino acid sequence. Results The proband was heterozygous for a known TSHR variant (p.Arg519His) and a novel frameshift TSHR variant (p.Val711Phefs*18), which removed 54 C-terminal residues and added a 17–amino acid frameshifted sequence. The loss of function of Val711Phefs*18-TSHR was confirmed in vitro, but the function of Val711*-TSHR was found to be normal. Western blotting showed the low protein expression of Val711Phefs*18-TSHR. Fusion of the frameshift sequence to green fluorescent protein or luciferase induced inactivation of them, indicating that the sequence acted as a degron. A systematic mutagenesis study revealed that the density of hydrophobic residues in the frameshift sequence determined the stability. Eight additional frameshift TSHR variants that covered all possible shifted frames in C-tail were created, and another frameshift variant (Thr748Profs*27) with similar effect was found. Conclusions We characterized a naturally occurring frameshift TSHR variant located in C-tail, and provided a unique evidence that hydrophobicity in the C-terminal region of the receptor affects protein stability.

scholarly journals A 49-residue sequence motif in the C terminus of Nav1.9 regulates trafficking of the channel to the plasma membrane

2019 ◽  
Vol 295 (4) ◽  
pp. 1077-1090 ◽  
Author(s):  
Daria V. Sizova ◽  
Jianying Huang ◽  
Elizabeth J. Akin ◽  
Mark Estacion ◽  
Carolina Gomis-Perez ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Recombinant Expression ◽  
Critical Role ◽  
Functional Expression ◽  
Hek293 Cells ◽  
Sequence Motif ◽  
Functional Studies ◽  
C Terminus ◽  
Mechanistic Basis ◽  
Heterologous Expression Systems

Genetic and functional studies have confirmed an important role for the voltage-gated sodium channel Nav1.9 in human pain disorders. However, low functional expression of Nav1.9 in heterologous systems (e.g. in human embryonic kidney 293 (HEK293) cells) has hampered studies of its biophysical and pharmacological properties and the development of high-throughput assays for drug development targeting this channel. The mechanistic basis for the low level of Nav1.9 currents in heterologous expression systems is not understood. Here, we implemented a multidisciplinary approach to investigate the mechanisms that govern functional Nav1.9 expression. Recombinant expression of a series of Nav1.9-Nav1.7 C-terminal chimeras in HEK293 cells identified a 49-amino-acid-long motif in the C terminus of the two channels that regulates expression levels of these chimeras. We confirmed the critical role of this motif in the context of a full-length channel chimera, Nav1.9-Ct49aaNav1.7, which displayed significantly increased current density in HEK293 cells while largely retaining the characteristic Nav1.9-gating properties. High-resolution live microscopy indicated that the newly identified C-terminal motif dramatically increases the number of channels on the plasma membrane of HEK293 cells. Molecular modeling results suggested that this motif is exposed on the cytoplasmic face of the folded C terminus, where it might interact with other channel partners. These findings reveal that a 49-residue-long motif in Nav1.9 regulates channel trafficking to the plasma membrane.

scholarly journals Importance of Branched-Chain Amino Acid Utilization in Francisella Intracellular Adaptation

2014 ◽  
Vol 83 (1) ◽  
pp. 173-183 ◽  
Author(s):  
Gael Gesbert ◽  
Elodie Ramond ◽  
Fabiola Tros ◽  
Julien Dairou ◽  
Eric Frapy ◽  
...  
Keyword(s):  
Amino Acid ◽  
Critical Role ◽  
Cell Types ◽  
Host Cells ◽  
Metabolic Adaptation ◽  
Amino Acid Transporters ◽  
Loss Of Function ◽  
Content Type ◽  
Branched Chain Amino Acid ◽  
Branched Chain

Intracellular bacterial pathogens have adapted their metabolism to optimally utilize the nutrients available in infected host cells. We recently reported the identification of an asparagine transporter required specifically for cytosolic multiplication ofFrancisella. In the present work, we characterized a new member of the major super family (MSF) of transporters, involved in isoleucine uptake. We show that this transporter (here designated IleP) plays a critical role in intracellular metabolic adaptation ofFrancisella. Inactivation of IleP severely impaired intracellularF. tularensissubsp.novicidamultiplication in all cell types tested and reduced bacterial virulence in the mouse model. To further establish the importance of theilePgene inF. tularensispathogenesis, we constructed a chromosomal deletion mutant ofileP(ΔFTL_1803) in theF. tularensissubsp.holarcticalive vaccine strain (LVS). Inactivation of IleP in theF. tularensisLVS provoked comparable intracellular growth defects, confirming the critical role of this transporter in isoleucine uptake. The data presented establish, for the first time, the importance of isoleucine utilization for efficient phagosomal escape and cytosolic multiplication ofFrancisellaand suggest that virulentF. tularensissubspecies have lost their branched-chain amino acid biosynthetic pathways and rely exclusively on dedicated uptake systems. This loss of function is likely to reflect an evolution toward a predominantly intracellular life style of the pathogen. Amino acid transporters should be thus considered major players in the adaptation of intracellular pathogens.

scholarly journals Werewolf, there wolf: variants in Hairless associated with hypotrichia and roaning in the lykoi cat breed

2020 ◽  
Author(s):  
Reuben M. Buckley ◽  
Barbara Gandolfi ◽  
Erica K. Creighton ◽  
Connor A. Pyne ◽  
Michelle L. LeRoy ◽  
...  
Keyword(s):  
Amino Acid ◽  
Hair Follicle ◽  
Stop Codon ◽  
Amino Acid Position ◽  
Lymphocytic Infiltration ◽  
Loss Of Function ◽  
Hair Coat ◽  
The Usa ◽  
Causal Variants ◽  
Sparse Hair

AbstractA variety of cat breeds have been developed via novelty selection on aesthetic, dermatological traits, such as coat colors and fur types. A recently developed breed, the lykoi, was bred from cats with a sparse hair coat with roaning, implying full color and all white hairs. The lykoi phenotype is a form of hypotrichia, presenting as significant reduction in the average numbers of follicles per hair follicle group as compared to domestic shorthair cats, a mild to severe perifollicular to mural lymphocytic infiltration in 77% of observed hair follicle groups, and the follicles are often miniaturized, dilated, and dysplastic. Whole genome sequencing was conducted on a single lykoi cat that was a cross between two independently ascertained lineages. Comparison to the 99 Lives dataset of 194 non-lykoi cats suggested two variants in the cat homolog for Hairless (HR: lysine demethylase and nuclear receptor corepressor) as candidate causal variants. The lykoi cat was a compound heterozygote for two loss of function variants in HR, an exon 3 c.1255_1256dupGT (chrB1:36040783), which should produce a stop codon at amino acid 420 (p.Gln420Serfs*100) and, an exon 18 c.3389insGACA (chrB1:36051555), which should produce a stop codon at amino acid position 1130 (p.Ser1130Argfs*29). Ascertainment of 14 additional cats from founder lineages from Canada, France and different areas of the USA identified four additional loss of function HR variants likely causing the highly similar phenotypic hair coat across the diverse cats. The novel variants in HR for cat hypotrichia can now be established between minor differences in the phenotypic presentations.

scholarly journals NBD2 Is Required for the Rescue of Mutant F508del CFTR by a Thiazole-Based Molecule: A Class II Corrector for the Multi-Drug Therapy of Cystic Fibrosis

Biomolecules ◽  
2021 ◽  
Vol 11 (10) ◽  
pp. 1417
Author(s):  
Chiara Brandas ◽  
Alessandra Ludovico ◽  
Alice Parodi ◽  
Oscar Moran ◽  
Enrico Millo ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Clinical Status ◽  
Anion Channel ◽  
Fluid Secretion ◽  
Functional Expression ◽  
Class Ii ◽  
Hek293 Cells ◽  
Loss Of Function ◽  
Epithelial Surface ◽  
Sites Of Action

Cystic fibrosis (CF) is caused by loss-of-function mutations in the CF transmembrane conductance regulator (CFTR) protein, an anion channel that regulates epithelial surface fluid secretion. The deletion of phenylalanine at position 508 (F508del) is the most common CFTR mutation. F508del CFTR is characterized by folding and trafficking defects, resulting in decreased functional expression of the protein on the plasma membrane. Several classes of small molecules, named correctors, have been developed to rescue defective F508del CFTR. Although individual correctors failed to improve the clinical status of CF patients carrying the F508del mutation, better results were obtained using correctors combinations. These results were obtained according to the premise that the administration of correctors having different sites of action should enhance F508del CFTR rescue. We investigated the putative site of action of an aminoarylthiazole 4-(3-chlorophenyl)-N-(3-(methylthio)phenyl)thiazol-2-amine, named FCG, with proven CFTR corrector activity, and its synergistic effect with the corrector VX809. We found that neither the total expression nor the maturation of WT CFTR transiently expressed in human embryonic kidney 293 cells was influenced by FCG, administrated alone or in combination with VX809. On the contrary, FCG was able to enhance F508del CFTR total expression, and its combination with VX809 provided a further effect, being able to increase not only the total expression but also the maturation of the mutant protein. Analyses on different CFTR domains and groups of domains, heterologously expressed in HEK293 cells, show that NBD2 is necessary for FCG corrector activity. Molecular modelling analyses suggest that FCG interacts with a putative region located into the NBD2, ascribing this molecule to class II correctors. Our study indicates that the continuous development and testing of combinations of correctors targeting different structural and functional defects of mutant CFTR is the best strategy to ensure a valuable therapeutic perspective to a larger cohort of CF patients.

scholarly journals Human rod photoreceptor cGMP-gated channel: amino acid sequence, gene structure, and functional expression

1992 ◽  
Vol 12 (8) ◽  
pp. 3248-3256 ◽  
Author(s):  
RS Dhallan ◽  
JP Macke ◽  
RL Eddy ◽  
TB Shows ◽  
RR Reed ◽  
...  
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Gene Structure ◽  
Functional Expression ◽  
Rod Photoreceptor ◽  
Gated Channel
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