Functional relevance of aromatic residues in the first transmembrane domain of P2X receptors

Oligodendrocytes facilitate rapid impulse propagation along the axons they myelinate and support their long-term integrity. However, the functional relevance of many myelin proteins has remained unknown. Here we find that expression of the tetraspan-transmembrane protein CMTM5 (Chemokine-like factor-like MARVEL-transmembrane domain containing protein 5) is highly enriched in oligodendrocytes and CNS myelin. Genetic disruption of the Cmtm5-gene in oligodendrocytes of mice does not impair the development or ultrastructure of CNS myelin. However, oligodendroglial Cmtm5-deficiency causes an early-onset progressive axonopathy, which we also observe in global and in tamoxifen-induced oligodendroglial Cmtm5-mutants. Presence of the Wlds mutation ameliorates the axonopathy, implying a Wallerian degeneration-like pathomechanism. These results indicate that CMTM5 is involved in the function of oligodendrocytes to maintain axonal integrity rather than myelin biogenesis.

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Photo-switchable tweezers illuminate pore-opening motions of an ATP-gated P2X ion channel

eLife ◽

10.7554/elife.11050 ◽

2016 ◽

Vol 5 ◽

Cited By ~ 24

Author(s):

Chloé Habermacher ◽

Adeline Martz ◽

Nicolas Calimet ◽

Damien Lemoine ◽

Laurie Peverini ◽

...

Keyword(s):

Bound States ◽

Transmembrane Domain ◽

P2x Receptors ◽

Structural Constraints ◽

Molecular Tweezers ◽

Gating Mechanism ◽

Outer Pore ◽

Pore Opening ◽

High Resolution Models ◽

Hinge Bending

P2X receptors function by opening a transmembrane pore in response to extracellular ATP. Recent crystal structures solved in apo and ATP-bound states revealed molecular motions of the extracellular domain following agonist binding. However, the mechanism of pore opening still remains controversial. Here we use photo-switchable cross-linkers as ‘molecular tweezers’ to monitor a series of inter-residue distances in the transmembrane domain of the P2X2 receptor during activation. These experimentally based structural constraints combined with computational studies provide high-resolution models of the channel in the open and closed states. We show that the extent of the outer pore expansion is significantly reduced compared to the ATP-bound structure. Our data further reveal that the inner and outer ends of adjacent pore-lining helices come closer during opening, likely through a hinge-bending motion. These results provide new insight into the gating mechanism of P2X receptors and establish a versatile strategy applicable to other membrane proteins.

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Membrane protein retention in the yeast Golgi apparatus: dipeptidyl aminopeptidase A is retained by a cytoplasmic signal containing aromatic residues.

The Journal of Cell Biology ◽

10.1083/jcb.121.6.1197 ◽

1993 ◽

Vol 121 (6) ◽

pp. 1197-1209 ◽

Cited By ~ 100

Author(s):

S F Nothwehr ◽

C J Roberts ◽

T H Stevens

Keyword(s):

Amino Acid ◽

Membrane Protein ◽

Transmembrane Domain ◽

Mutational Analysis ◽

Vacuolar Membrane ◽

Type Ii ◽

Golgi Membrane ◽

Aromatic Residues ◽

Golgi Retention ◽

Dipeptidyl Aminopeptidase

The mechanism by which yeast dipeptidyl aminopeptidase (DPAP) A, type II integral membrane protein, is retained in the late Golgi apparatus has been investigated. Prior work demonstrated that the 118-amino acid cytoplasmic domain is both necessary and sufficient for Golgi retention and that mutant or overexpressed DPAP A no longer retained in the Golgi was delivered directly to the vacuolar membrane (Roberts, C. J., S. F. Nothwehr, and T. H. Stevens. 1992. J. Cell Biol. 119:69-83). Replacement of the DPAP A transmembrane domain with a synthetic hydrophobic sequence did not affect either Golgi retention of DPAP A or vacuolar delivery of the retention-defective form of DPAP A. These results indicate that the DPAP A transmembrane domain is not involved in either Golgi retention or targeting of this membrane protein. A detailed mutational analysis of the cytoplasmic domain of DPAP A indicated that the most important elements for retention were within the eight residue stretch 85-92. A 10-amino acid region from DPAP A (81-90) was sufficient for Golgi retention of alkaline phosphatase, a type II vacuolar membrane protein. Detailed mutational analysis within this 10-amino acid sufficient region demonstrated that a Phe-X-Phe-X-Asp motif was absolutely required for efficient retention. The efficiency of Golgi retention via the DPAP A signal could be diminished by overexpression of wild type but not retention-defective versions of Kex2p, another late Golgi membrane protein, suggesting that multiple Golgi membrane proteins may be retained by a common machinery. These results imply a role for a cytoplasmic signal involving aromatic residues in retention of late Golgi membrane proteins in the yeast Saccharomyces cerevisiae.

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Aromatic Amino Acids in the Juxtamembrane Domain of Severe Acute Respiratory Syndrome Coronavirus Spike Glycoprotein Are Important for Receptor-Dependent Virus Entry and Cell-Cell Fusion

Journal of Virology ◽

10.1128/jvi.01805-07 ◽

2008 ◽

Vol 82 (6) ◽

pp. 2883-2894 ◽

Cited By ~ 25

Author(s):

Megan W. Howard ◽

Emily A. Travanty ◽

Scott A. Jeffers ◽

M. K. Smith ◽

Sonia T. Wennier ◽

...

Keyword(s):

Amino Acids ◽

Cell Fusion ◽

Transmembrane Domain ◽

Virus Entry ◽

Aromatic Amino Acids ◽

Wild Type ◽

Aromatic Residues ◽

Wild Type Level ◽

S Proteins ◽

Cell Cell

ABSTRACT The severe acute respiratory syndrome coronavirus (SARS-CoV) spike glycoprotein (S) is a class I viral fusion protein that binds to its receptor glycoprotein, human angiotensin converting enzyme 2 (hACE2), and mediates virus entry and cell-cell fusion. The juxtamembrane domain (JMD) of S is an aromatic amino acid-rich region proximal to the transmembrane domain that is highly conserved in all coronaviruses. Alanine substitutions for one or two of the six aromatic residues in the JMD did not alter the surface expression of the SARS-CoV S proteins with a deletion of the C-terminal 19 amino acids (S Δ19) or reduce binding to soluble human ACE2 (hACE2). However, hACE2-dependent entry of trypsin-treated retrovirus pseudotyped viruses expressing JMD mutant S Δ19 proteins was greatly reduced. Single alanine substitutions for aromatic residues reduced entry to 10 to 60% of the wild-type level. The greatest reduction was caused by residues nearest the transmembrane domain. Four double alanine substitutions reduced entry to 5 to 10% of the wild-type level. Rapid hACE2-dependent S-mediated cell-cell fusion was reduced to 60 to 70% of the wild-type level for all single alanine substitutions and the Y1188A/Y1191A protein. S Δ19 proteins with other double alanine substitutions reduced cell-cell fusion further, from 40% to less than 20% of wild-type levels. The aromatic amino acids in the JMD of the SARS-CoV S glycoprotein play critical roles in receptor-dependent virus-cell and cell-cell fusion. Because the JMD is so highly conserved in all coronavirus S proteins, it is a potential target for development of drugs that may inhibit virus entry and/or cell-cell fusion mediated by S proteins of all coronaviruses.

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The roles of aromatic residues in the glycine receptor transmembrane domain

BMC Neuroscience ◽

10.1186/s12868-018-0454-8 ◽

2018 ◽

Vol 19 (1) ◽

Cited By ~ 6

Author(s):

Bijun Tang ◽

Sarah C. R. Lummis

Keyword(s):

Transmembrane Domain ◽

Glycine Receptor ◽

Aromatic Residues

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Roles of conserved ectodomain cysteines of the rat P2X4 purinoreceptor in agonist binding and channel gating

Physiological Research ◽

10.33549/physiolres.931979 ◽

2010 ◽

pp. 927-935

Author(s):

MB Rokic ◽

V Tvrdoňová ◽

V Vávra ◽

M Jindřichová ◽

T Obšil ◽

...

Keyword(s):

Disulfide Bonds ◽

Response Pattern ◽

Transmembrane Domain ◽

Binding Pocket ◽

P2x Receptors ◽

Hek293 Cells ◽

Receptor Function ◽

Agonist Binding ◽

P2x4 Receptor ◽

Maximum Current

Mammalian P2X receptors contain 10 conserved cysteine residues in their ectodomains, which form five disulfide bonds (SS1-5). Here, we analyzed the relevance of these disulfide pairs in rat P2X4 receptor function by replacing one or both cysteines with alanine or threonine, expressing receptors in HEK293 cells and studying their responsiveness to ATP in the absence and presence of ivermectin, an allostenic modulator of these channels. Response to ATP was not altered when both cysteines forming the SS3 bond (C132-C159) were replaced with threonines. Replacement of SS1 (C116-C165), SS2 (C126-C149) and SS4 (C217-C227), but not SS5 (C261-C270), cysteine pairs with threonines resulted in decreased sensitivity to ATP and faster deactivation times. The maximum current amplitude was reduced in SS2, SS4 and SS5 double mutants and could be partially rescued by ivermectin in SS2 and SS5 double mutants. This response pattern was also observed in numerous single residue mutants, but receptor function was not affected when the 217 cysteine was replaced with threonine or arginine or when the 261 cysteine was replaced with alanine. These results suggest that the SS1, SS2 and SS4 bonds contribute substantially to the structure of the ligand binding pocket, while the SS5 bond located towards the transmembrane domain contributes to receptor gating.

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Functional relevance of cortical plasticity demonstrated with transcranial magnetic stimulation (TMS)

Electroencephalography and Clinical Neurophysiology ◽

10.1016/s0013-4694(97)87999-4 ◽

1997 ◽

Vol 103 (1) ◽

pp. 21 ◽

Cited By ~ 2

Author(s):

L Cohen

Keyword(s):

Transcranial Magnetic Stimulation ◽

Magnetic Stimulation ◽

Cortical Plasticity ◽

Functional Relevance

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Assessing Stressors Related to Migration in Patients With Psychiatric Disorders

European Journal of Psychological Assessment ◽

10.1027/1015-5759/a000106 ◽

2012 ◽

Vol 28 (4) ◽

pp. 262-269 ◽

Cited By ~ 5

Author(s):

Matthias Johannes Müller ◽

Suzan Kamcili-Kubach ◽

Songül Strassheim ◽

Eckhardt Koch

Keyword(s):

Interrater Reliability ◽

Item Analysis ◽

Global Assessment Of Functioning ◽

Family Conflicts ◽

Response Options ◽

Reliability Indices ◽

Dsm Iv ◽

Functional Relevance ◽

Sum Scores ◽

Gaf Scores

A 10-item instrument for the assessment of probable migration-related stressors was developed based on previous work (MIGSTR10) and interrater reliability was tested in a chart review study. The MIGSTR10 and nine nonspecific stressors of the DSM-IV Axis IV (DSMSTR9) were put into a questionnaire format with categorical and dimensional response options. Charts of 100 inpatients (50 Turkish migrants [MIG], 50 native German patients [CON]) with affective or anxiety disorder were reviewed by three independent raters and MIGSTR10, DSMSTR9, and Global Assessment of Functioning scale (GAF) scores were obtained. Interrater reliability indices (ICC) of items and sum scores were calculated. The prevalence of single migration-related stressors in MIG ranged from 15% to 100% (CON 0–92%). All items of the MIGSTR10 (ICC 0.58–0.92) and the DSMSTR9 (ICC 0.56–0.96) reached high to very high interrater agreement (p < .0005). The item analysis of the MIGSTR10 revealed sufficient internal consistency (Cronbach’s α = 0.68/0.69) and only one item (“family conflicts”) without substantial correlation with the remaining scale. Correlation analyses showed a significant overlap of dimensional MIGSTR10 scores (r² = 0.25; p < .01) and DSMSTR9 scores (r² = 9%; p < .05) with GAF scores in MIG indicating functional relevance. MIGSTR10 is considered a feasible, economic, and reliable instrument for the assessment of stressors potentially related to migration.

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Recombinant Human Soluble Thrombomodulin Delivers Bounded Thrombin to Antithrombin III: Thrombomodulin Associates with Free Thrombin and Is Recycled to Activate Protein C

Thrombosis and Haemostasis ◽

10.1055/s-0038-1649597 ◽

1993 ◽

Vol 70 (03) ◽

pp. 418-422 ◽

Cited By ~ 23

Author(s):

Masaharu Aritomi ◽

Naoko Watanabe ◽

Rika Ohishi ◽

Komakazu Gomi ◽

Takao Kiyota ◽

...

Keyword(s):

Protein C ◽

Antithrombin Iii ◽

Transmembrane Domain ◽

Chinese Hamster Ovary Cells ◽

Time Lag ◽

Chinese Hamster ◽

Ovary Cells ◽

Soluble Thrombomodulin ◽

Simulation Based ◽

Recombinant Human Soluble Thrombomodulin

SummaryRecombinant human soluble thrombomodulin (rhs-TM), having no transmembrane domain or chondroitin sulfate, was expressed in Chinese hamster ovary cells. Interactions between rhs-TM, thrombin (Th), protein C (PC) and antithrombin III (ATIII) were studied. Equilibrium between rhs-TM and Th had no detectable time lag in clotting inhibition (K d = 26 nM) or PC activation (K d = 22 nM), while ATIII inhibited Th at a bimolecular rate constant = 5,200 M-1s-1 (K d <0.2 nM). A mixture of ATIII, Th and rhs-TM showed that ATIII reacted with Th slower than rhs-TM, whose presence did not affect the reaction between ATIII and Th. In a mixture of rhs-TM, ATIII and PC, the repeated addition of Th caused the repeated activation of PC; which was consistent with the Simulation based on the assumption that rhs-TM is recycled as a Th cofactor. From these results, we concluded that upon inhibition of the rhs-TM-Th complex by ATIII, rhs-TM is released to recombine with free Th and begins to activate PC, while the Th-ATIII complex does not affect rhs-TM-Th equilibrium.

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