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Calibration and functional analysis of three genetically encoded Cl(-)/pH sensors

Mukhtarov, M ; Liguori, L ; Waseem, T ; Rocca, F ; Buldakova, S ; Arosio, D ; Bregestovski, P

Frontiers in molecular neuroscience, 2013-01, Vol.6, p.9-9 [Periódico revisado por pares]

Switzerland: Frontiers Research Foundation

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  • Título:
    Calibration and functional analysis of three genetically encoded Cl(-)/pH sensors
  • Autor: Mukhtarov, M ; Liguori, L ; Waseem, T ; Rocca, F ; Buldakova, S ; Arosio, D ; Bregestovski, P
  • Assuntos: Binding sites ; Biophysics ; Biosensors ; Brain research ; Chloride ; Deoxyribonucleic acid ; DNA ; E coli ; Enzymatic activity ; fluorescent bioSensors ; Fusion protein ; Hydrogen ; Intracellular ; intracellular chloride ; intracellular pH ; Mutagenesis ; Mutation ; Neurons ; Neuroscience ; Neurosciences ; non-invasive monitoring ; optogenetics ; pH effects ; Plasmids ; Proteins ; Receptor channels ; Sensors
  • É parte de: Frontiers in molecular neuroscience, 2013-01, Vol.6, p.9-9
  • Notas: ObjectType-Article-1
    SourceType-Scholarly Journals-1
    ObjectType-Feature-2
    content type line 23
    Reviewed by: Colin J. Akerman, University of Oxford Academic Research Fellow, UK; Alexandre Mourot, Université Pierre et Marie Curie, France
    Edited by: Pau Gorostiza, Catalan Institution for Research and Advanced Studies, Spain
  • Descrição: Monitoring of the intracellular concentrations of Cl(-) and H(+) requires sensitive probes that allow reliable quantitative measurements without perturbation of cell functioning. For these purposes the most promising are genetically encoded fluorescent biosensors, which have become powerful tools for non-invasive intracellular monitoring of ions, molecules, and enzymatic activity. A ratiometric CFP/YFP-based construct with a relatively good sensitivity to Cl(-) has been developed (Markova et al., 2008; Waseem et al., 2010). Recently, a combined Cl(-)/pH sensor (ClopHensor) opened the way for simultaneous ratiometric measurement of these two ions (Arosio et al., 2010). ClopHensor was obtained by fusion of a red-fluorescent protein (DsRed-monomer) to the E(2)GFP variant that contains a specific Cl(-)-binding site. This construct possesses pK a = 6.8 for H(+) and K d in the 40-50 mM range for Cl(-) at physiological pH (~7.3). As in the majority of cell types the intracellular Cl(-) concentration ([Cl(-)] i ) is about 10 mM, the development of sensors with higher sensitivity is highly desirable. Here, we report the intracellular calibration and functional characterization of ClopHensor and its two derivatives: the membrane targeting PalmPalm-ClopHensor and the H148G/V224L mutant with improved Cl(-) affinity, reduced pH dependence, and pK a shifted to more alkaline values. For functional analysis, constructs were expressed in CHO cells and [Cl(-)] i was changed by using pipettes with different Cl(-) concentrations during whole-cell recordings. K d values for Cl(-) measured at 33°C and pH ~7.3 were, respectively, 39, 47, and 21 mM for ClopHensor, PalmPalm-ClopHensor, and the H148G/V224L mutant. PalmPalm-ClopHensor resolved responses to activation of Cl(-)-selective glycine receptor (GlyR) channels better than did ClopHensor. Our observations indicate that these different ClopHensor constructs are promising tools for non-invasive measurement of [Cl(-)] i in various living cells.
  • Editor: Switzerland: Frontiers Research Foundation
  • Idioma: Inglês
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  • Realização: ABCD-USP USP   Logos de Redes Sociais: Freepik

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