Structural determinants of oligomerization of the aquaporin-4 channel
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Abstract©2016 by The American Society for Biochemistry and Molecular Biology, Inc. The aquaporin (AQP) family of integral membrane protein channels mediate cellular water and solute flow. Although qualitative and quantitative differences in channel permeability, selectivity, subcellular localization, and trafficking responses have been observed for different members of the AQP family, the signature homotetrameric quaternary structure is conserved. Using a variety of biophysical techniques, we show that mutations to an intracellular loop (loop D) of human AQP4 reduce oligomerization. Non-tetrameric AQP4 mutants are unable to relocalize to the plasma membrane in response to changes in extracellular tonicity, despite equivalent constitutive surface expression levels and water permeability to wild-type AQP4. A network of AQP4 loop D hydrogen bonding interactions, identified using molecular dynamics simulations and based on a comparative mutagenic analysis of AQPs 1, 3, and 4, suggest that loop D interactions may provide a general structural framework for tetrameric assembly within theAQPfamily.
CitationKitchen, P., Conner, M. T., Bill, R. M. and Conner, A. C. (2016) Structural determinants of oligomerization of the aquaporin-4 channel, Journal of Biological Chemistry, 29(13), pp. 6858-6871.
JournalJournal of Biological Chemistry
SponsorsSupported by Biotechnology and Biological Sciences Research Council Grants BB/I019960/1, BB/K013319/1, and BB/L502194/1 and Innovative Medicines Joint Undertaking under Grant Agreement 115583 to the ND4BB ENABLE Consortium.
Except where otherwise noted, this item's license is described as Licence for published version: Creative Commons Attribution 4.0 International
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Plasma membrane abundance of human aquaporin 5 is dynamically regulated by multiple pathwaysKitchen, P; Öberg, F; Sjöhamn, J; Hedfalk, K; Bill, RM; Törnroth-Horsefield, S; Conner, Matthew T.; Conner, Alex C.; Molecular Organization and Assembly in Cells Doctoral Training Centre, University of Warwick, Coventry, United Kingdom. (Public Library of Science (PLoS), 2015-11-16)© 2015 Kitchen et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Aquaporin membrane protein channels mediate cellular water flow. Human aquaporin 5 (AQP5) is highly expressed in the respiratory system and secretory glands where it facilitates the osmotically-driven generation of pulmonary secretions, saliva, sweat and tears. Dysfunctional trafficking of AQP5 has been implicated in several human disease states, including Sjögren's syndrome, bronchitis and cystic fibrosis. In order to investigate how the plasma membrane expression levels of AQP5 are regulated, we studied real-time translocation of GFP-tagged AQP5 in HEK293 cells. We show that AQP5 plasma membrane abundance in transfected HEK293 cells is rapidly and reversibly regulated by at least three independent mechanisms involving phosphorylation at Ser156, protein kinase A activity and extracellular tonicity. The crystal structure of a Ser156 phosphomimetic mutant indicates that its involvement in regulating AQP5 membrane abundance is not mediated by a conformational change of the carboxy-terminus. We suggest that together these pathways regulate cellular water flow.
Cell-Penetrating PeptidesHowl, John; Jones, Sarah (2015)The multi-domain architecture of many human proteins provides a structural basis for the physical maintenance of interactomes, or networks of protein-protein interactions (PPIs), that are so obviously crucial to cellular functions. Moreover, the structural and electrostatic complementarity provided by PPI interfaces, predominantly located on protein surfaces, is a fundamental component of signal transduction events that are known to be compromised in human diseases including many cancers.The pharmacokinetic advantages provided by cell-penetrating peptides (CPPs) are entirely compatible with the development of intrinsically permeable agents capable of modulating intracellular PPIs. Thus, the term bioportide is a useful descriptor of numerous bioactive CPPs that are distinct from the more usual inert CPP vectors. Herein we illustrate a generic strategy, predominantly centered upon the identification of cationic peptides derived from helical protein domains, which offers a reliable platform to identify bioportides capable of modulating intracellular signal transduction events. In addition, we describe robust methodologies to determine the precise intracellular distribution of fluorescent bioportides and present assays routinely employed to screen for the detrimental pharmacodynamic properties often exhibited by both CPPs and bioportides; namely adverse cytotoxicity and the receptor-independent stimulation of mast cell secretion.
Identification and molecular mechanisms of the rapid tonicity-induced relocalization of the aquaporin 4 channelKitchen, P; Day, RE; Taylor, LHJ; Salman, MM; Bill, RM; Conner, Matthew T.; Conner, Alex C.; From the Molecular Organisation and Assembly in Cells Doctoral Training Centre, University of Warwick, Coventry CV4 7AL. (American Society for Biochemistry & Molecular Biology (ASBMB), 2015-05-26)© 2015 by The American Society for Biochemistry and Molecular Biology, Inc. Background: The water channel protein aquaporin 4 (AQP4) controls water permeability of the blood-brain barrier. Results: Hypotonicity induces rapid relocalization of AQP4 in a calcium-, calmodulin-, and kinase-dependent manner. Conclusion: AQP4 can be relocalized between the cell membrane and intracellular compartments. Significance: Pharmacological modulation of AQP4 membrane localization could provide a new approach to treating brain edema.