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Pore dilation occurs in TRPA1 but not in TRPM8 channels
Jun Chen, Donghee Kim, Bruce R Bianchi, Eric J Cavanaugh, Connie R Faltynek, Philip R Kym, Regina M Reilly
Molecular Pain , 2009, DOI: 10.1186/1744-8069-5-3
Abstract: Abundantly expressed in sensory neurons, TRPV1, TRPA1 and TRPM8 are involved in sensory function, pain and neurogenic inflammation [1]. The function of these ion channels has been attributed to their ability to pass certain ion species across the plasma membrane. Once activated, TRPV1, TRPA1 and TRPM8 are permeable to small cations such as Ca2+, K+, Na+; hence, channel activation simultaneously depolarizes the plasma membrane and raises intracellular Ca2+, which subsequently triggers a variety of physiological processes. By analogy to voltage-gated K+ channels, it is assumed that ion selectivity of TRP channels should be an invariant signature to the respective channel. However, this notion has been challenged recently. When activated, TRPV1 exhibits time and agonist-dependent changes in ion selectivity [2]. In fact, TRPV1 undergoes pore dilation and allows permeation of large organic cations, including spermine (202.3 Da), NMDG (195.2 Da), Yo-Pro (376 Da), gentamycin (477.6 Da) and QX-314 [3-7]. Here we explored whether TRPA1 and TRPM8 undergo pore dilation by examining Yo-Pro uptake and changes in ion selectivity upon channel activation.Yo-Pro is a divalent cation impermeable to the plasma membrane. However, under certain conditions, it can enter cells, bind nucleic acids and emit fluorescence. Hence the uptake of Yo-Pro has been used previously as an indicator of pore dilation [2,8,9]. In HEK293-F cells transiently expressing rat TRPA1, allyl isothiocyanate (AITC) evoked robust increases in intracellular Ca2+ (Fig. 1A). Concomitantly, AITC also induced Yo-Pro uptake in a concentration-dependent manner (Fig. 1B). At higher concentrations of AITC (100 or 300 μM), the increase in fluorescence was immediately noticeable and continued to increase for about 50 min. In addition, AITC also induced Ca2+ influx and Yo-Pro uptake in cells expressing human TRPA1 and mouse TRPA1, but not in untransfected cells (data not shown). In cells expressing human TRPM8, menthol activat
Single Residues in the Outer Pore of TRPV1 and TRPV3 Have Temperature-Dependent Conformations  [PDF]
Sung Eun Kim, Ardem Patapoutian, J?rg Grandl
PLOS ONE , 2013, DOI: 10.1371/journal.pone.0059593
Abstract: Thermosensation is mediated by ion channels that are highly temperature-sensitive. Several members of the family of transient receptor potential (TRP) ion channels are activated by cold or hot temperatures and have been shown to function as temperature sensors in vivo. The molecular mechanism of temperature-sensitivity of these ion channels is not understood. A number of domains or even single amino acids that regulate temperature-sensitivity have been identified in several TRP channels. However, it is unclear what precise conformational changes occur upon temperature activation. Here, we used the cysteine accessibility method to probe temperature-dependent conformations of single amino acids in TRP channels. We screened over 50 amino acids in the predicted outer pore domains of the heat-activated ion channels TRPV1 and TRPV3. In both ion channels we found residues that have temperature-dependent accessibilities to the extracellular solvent. The identified residues are located within the second predicted extracellular pore loop. These residues are identical or proximal to residues that were shown to be specifically required for temperature-activation, but not chemical activation. Our data precisely locate conformational changes upon temperature-activation within the outer pore domain. Collectively, this suggests that these specific residues and the second predicted pore loop in general are crucial for the temperature-activation mechanism of these heat-activated thermoTRPs.
Cholesterol Depletion Inactivates XMRV and Leads to Viral Envelope Protein Release from Virions: Evidence for Role of Cholesterol in XMRV Infection  [PDF]
Yuyang Tang, Alvin George, Thyneice Taylor, James E. K. Hildreth
PLOS ONE , 2012, DOI: 10.1371/journal.pone.0048013
Abstract: Membrane cholesterol plays an important role in replication of HIV-1 and other retroviruses. Here, we report that the gammaretrovirus XMRV requires cholesterol and lipid rafts for infection and replication. We demonstrate that treatment of XMRV with a low concentration (10 mM) of 2-hydroxypropyl-β-cyclodextrin (2OHpβCD) partially depleted virion-associated cholesterol resulting in complete inactivation of the virus. This effect could not be reversed by adding cholesterol back to treated virions. Further analysis revealed that following cholesterol depletion, virus-associated Env protein was significantly reduced while the virions remained intact and retained core proteins. Increasing concentrations of 2OHpβCD (≥20 mM) resulted in loss of the majority of virion-associated cholesterol, causing disruption of membrane integrity and loss of internal Gag proteins and viral RNA. Depletion of cholesterol from XMRV-infected cells significantly reduced virus release, suggesting that cholesterol and intact lipid rafts are required for the budding process of XMRV. These results suggest that unlike glycoproteins of other retroviruses, the association of XMRV glycoprotein with virions is highly dependent on cholesterol and lipid rafts.
Conformational Changes Underlying Pore Dilation in the Cytoplasmic Domain of Mammalian Inward Rectifier K+ Channels  [PDF]
Atsushi Inanobe, Atsushi Nakagawa, Yoshihisa Kurachi
PLOS ONE , 2013, DOI: 10.1371/journal.pone.0079844
Abstract: The cytoplasmic domain of inward rectifier K+ (Kir) channels associates with cytoplasmic ligands and undergoes conformational change to control the gate present in its transmembrane domain. Ligand-operated activation appears to cause dilation of the pore at the cytoplasmic domain. However, it is still unclear how the cytoplasmic domain supports pore dilation and how alterations to this domain affect channel activity. In the present study, we focused on 2 spatially adjacent residues, i.e., Glu236 and Met313, of the G protein-gated Kir channel subunit Kir3.2. In the closed state, these pore-facing residues are present on adjacent βD and βH strands, respectively. We mutated both residues, expressed them with the m2-muscarinic receptor in Xenopus oocytes, and measured the acetylcholine-dependent K+ currents. The dose-response curves of the Glu236 mutants tended to be shifted to the right. In comparison, the slopes of the concentration-dependent curves were reduced and the single-channel properties were altered in the Met313 mutants. The introduction of arginine at position 236 conferred constitutive activity and caused a leftward shift in the conductance-voltage relationship. The crystal structure of the cytoplasmic domain of the mutant showed that the arginine contacts the main chains of the βH and βI strands of the adjacent subunit. Because the βH strand forms a β sheet with the βI and βD strands, the immobilization of the pore-forming β sheet appears to confer unique properties to the mutant. These results suggest that the G protein association triggers pore dilation at the cytoplasmic domain in functional channels, and the pore-constituting structural elements contribute differently to these conformational changes.
Cholesterol Depletion Disorganizes Oocyte Membrane Rafts Altering Mouse Fertilization  [PDF]
Jorgelina Buschiazzo, Come Ialy-Radio, Jana Auer, Jean-Philippe Wolf, Catherine Serres, Brigitte Lefèvre, Ahmed Ziyyat
PLOS ONE , 2013, DOI: 10.1371/journal.pone.0062919
Abstract: Drastic membrane reorganization occurs when mammalian sperm binds to and fuses with the oocyte membrane. Two oocyte protein families are essential for fertilization, tetraspanins and glycosylphosphatidylinositol-anchored proteins. The firsts are associated to tetraspanin-enriched microdomains and the seconds to lipid rafts. Here we report membrane raft involvement in mouse fertilization assessed by cholesterol modulation using methyl-β-cyclodextrin. Cholesterol removal induced: (1) a decrease of the fertilization rate and index; and (2) a delay in the extrusion of the second polar body. Cholesterol repletion recovered the fertilization ability of cholesterol-depleted oocytes, indicating reversibility of these effects. In vivo time-lapse analyses using fluorescent cholesterol permitted to identify the time-point at which the probe is mainly located at the plasma membrane enabling the estimation of the extent of the cholesterol depletion. We confirmed that the mouse oocyte is rich in rafts according to the presence of the raft marker lipid, ganglioside GM1 on the membrane of living oocytes and we identified the coexistence of two types of microdomains, planar rafts and caveolae-like structures, by terms of two differential rafts markers, flotillin-2 and caveolin-1, respectively. Moreover, this is the first report that shows characteristic caveolae-like invaginations in the mouse oocyte identified by electron microscopy. Raft disruption by cholesterol depletion disturbed the subcellular localization of the signal molecule c-Src and the inhibition of Src kinase proteins prevented second polar body extrusion, consistent with a role of Src-related kinases in fertilization via signaling complexes. Our data highlight the functional importance of intact membrane rafts for mouse fertilization and its dependence on cholesterol.
More Than a Pore: The Cellular Response to Cholesterol-Dependent Cytolysins  [PDF]
Sara K. B. Cassidy,Mary X. D. O'Riordan
Toxins , 2013, DOI: 10.3390/toxins5040618
Abstract: Targeted disruption of the plasma membrane is a ubiquitous form of attack used in all three domains of life. Many bacteria secrete pore-forming proteins during infection with broad implications for pathogenesis. The cholesterol-dependent cytolysins (CDC) are a family of pore-forming toxins expressed predominately by Gram-positive bacterial pathogens. The structure and assembly of some of these oligomeric toxins on the host membrane have been described, but how the targeted cell responds to intoxication by the CDCs is not as clearly understood. Many CDCs induce lysis of their target cell and can activate apoptotic cascades to promote cell death. However, the extent to which intoxication causes cell death is both CDC- and host cell-dependent, and at lower concentrations of toxin, survival of intoxicated host cells is well documented. Additionally, the effect of CDCs can be seen beyond the plasma membrane, and it is becoming increasingly clear that these toxins are potent regulators of signaling and immunity, beyond their role in intoxication. In this review, we discuss the cellular response to CDC intoxication with emphasis on the effects of pore formation on the host cell plasma membrane and subcellular organelles and whether subsequent cellular responses contribute to the survival of the affected cell.
Effect of Plasma Membrane Cholesterol Depletion on Glucose Transport Regulation in Leukemia Cells  [PDF]
Cristiana Caliceti, Laura Zambonin, Cecilia Prata, Francesco Vieceli Dalla Sega, Gabriele Hakim, Silvana Hrelia, Diana Fiorentini
PLOS ONE , 2012, DOI: 10.1371/journal.pone.0041246
Abstract: GLUT1 is the predominant glucose transporter in leukemia cells, and the modulation of glucose transport activity by cytokines, oncogenes or metabolic stresses is essential for their survival and proliferation. However, the molecular mechanisms allowing to control GLUT1 trafficking and degradation are still under debate. In this study we investigated whether plasma membrane cholesterol depletion plays a role in glucose transport activity in M07e cells, a human megakaryocytic leukemia line. To this purpose, the effect of cholesterol depletion by methyl-β-cyclodextrin (MBCD) on both GLUT1 activity and trafficking was compared to that of the cytokine Stem Cell Factor (SCF). Results show that, like SCF, MBCD led to an increased glucose transport rate and caused a subcellular redistribution of GLUT1, recruiting intracellular transporter molecules to the plasma membrane. Due to the role of caveolae/lipid rafts in GLUT1 stimulation in response to many stimuli, we have also investigated the GLUT1 distribution along the fractions obtained after non ionic detergent treatment and density gradient centrifugation, which was only slightly changed upon MBCD treatment. The data suggest that MBCD exerts its action via a cholesterol-dependent mechanism that ultimately results in augmented GLUT1 translocation. Moreover, cholesterol depletion triggers GLUT1 translocation without the involvement of c-kit signalling pathway, in fact MBCD effect does not involve Akt and PLCγ phosphorylation. These data, together with the observation that the combined MBCD/SCF cell treatment caused an additive effect on glucose uptake, suggest that the action of SCF and MBCD may proceed through two distinct mechanisms, the former following a signalling pathway, and the latter possibly involving a novel cholesterol dependent mechanism.
A New Model for Pore Formation by Cholesterol-Dependent Cytolysins  [PDF]
Cyril F. Reboul,James C. Whisstock,Michelle A. Dunstone
PLOS Computational Biology , 2014, DOI: doi/10.1371/journal.pcbi.1003791
Abstract: Cholesterol Dependent Cytolysins (CDCs) are important bacterial virulence factors that form large (200–300 ?) membrane embedded pores in target cells. Currently, insights from X-ray crystallography, biophysical and single particle cryo-Electron Microscopy (cryo-EM) experiments suggest that soluble monomers first interact with the membrane surface via a C-terminal Immunoglobulin-like domain (Ig; Domain 4). Membrane bound oligomers then assemble into a prepore oligomeric form, following which the prepore assembly collapses towards the membrane surface, with concomitant release and insertion of the membrane spanning subunits. During this rearrangement it is proposed that Domain 2, a region comprising three β-strands that links the pore forming region (Domains 1 and 3) and the Ig domain, must undergo a significant yet currently undetermined, conformational change. Here we address this problem through a systematic molecular modeling and structural bioinformatics approach. Our work shows that simple rigid body rotations may account for the observed collapse of the prepore towards the membrane surface. Support for this idea comes from analysis of published cryo-EM maps of the pneumolysin pore, available crystal structures and molecular dynamics simulations. The latter data in particular reveal that Domains 1, 2 and 4 are able to undergo significant rotational movements with respect to each other. Together, our data provide new and testable insights into the mechanism of pore formation by CDCs.
The Cholesterol-Dependent Cytolysin Signature Motif: A Critical Element in the Allosteric Pathway that Couples Membrane Binding to Pore Assembly  [PDF]
Kelley J. Dowd,Rodney K. Tweten
PLOS Pathogens , 2012, DOI: 10.1371/journal.ppat.1002787
Abstract: The cholesterol-dependent cytolysins (CDCs) constitute a family of pore-forming toxins that contribute to the pathogenesis of a large number of Gram-positive bacterial pathogens.The most highly conserved region in the primary structure of the CDCs is the signature undecapeptide sequence (ECTGLAWEWWR). The CDC pore forming mechanism is highly sensitive to changes in its structure, yet its contribution to the molecular mechanism of the CDCs has remained enigmatic. Using a combination of fluorescence spectroscopic methods we provide evidence that shows the undecapeptide motif of the archetype CDC, perfringolysin O (PFO), is a key structural element in the allosteric coupling of the cholesterol-mediated membrane binding in domain 4 (D4) to distal structural changes in domain 3 (D3) that are required for the formation of the oligomeric pore complex. Loss of the undecapeptide function prevents all measurable D3 structural transitions, the intermolecular interaction of membrane bound monomers and the assembly of the oligomeric pore complex. We further show that this pathway does not exist in intermedilysin (ILY), a CDC that exhibits a divergent undecapeptide and that has evolved to use human CD59 rather than cholesterol as its receptor. These studies show for the first time that the undecapeptide of the cholesterol-binding CDCs forms a critical element of the allosteric pathway that controls the assembly of the pore complex.
Increased basolateral sorting of carcinoembryonic antigen in a polarized colon carcinoma cell line after cholesterol depletion-Implications for treatment of inflammatory bowel disease  [cached]
Robert Ehehalt, Markus Krautter, Martin Zorn, Richard Sparla, Joachim Füllekrug, Hasan Kulaksiz, Wolfgang Stremmel
World Journal of Gastroenterology , 2008,
Abstract: AIM: To investigate a possible increase of basolateral expression of carcinoembryonic antigen (CEA) by interfering with the apical transport machinery, we studied the effect of cholesterol depletion on CEA sorting and secretion.METHODS: Cholesterol depletion was performed in polarized Caco-2 cells using lovastatin and methyl-β-cyclodextrin.RESULTS: We show that CEA is predominantly expressed and secreted at the apical surface. Reduction of the cholesterol level of the cell by 40%-50% with lovastatin and methyl-β-cyclodextrin led to a significant change of the apical-to-basolateral transport ratio towards the basolateral membrane.CONCLUSION: As basolateral expression of CEA has been suggested to have anti-inflammatory properties, Cholesterol depletion of enterocytes might be a potential approach to influence the course of inflammatory bowel disease.
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