title: Synchronous Bioimaging of Intracellular pH and Chloride Based on LSS Fluorescent Protein.
abstract: Ion homeostasis regulates critical physiological processes in the living cell. Intracellular chloride concentration not only contributes in setting the membrane potential of quiescent cells but it also plays a role in modulating the dynamic voltage changes during network activity. Dynamic chloride imaging demands new tools, allowing faster acquisition rates and correct accounting of concomitant pH changes. Joining a long-Stokes-shift red-fluorescent protein to a GFP variant with high sensitivity to pH and chloride, we obtained LSSmClopHensor, a genetically encoded fluorescent biosensor optimized for the simultaneous chloride and pH imaging and requiring only two excitation wavelengths (458 and 488 nm). LSSmClopHensor allowed us to monitor the dynamic changes of intracellular pH and chloride concentration during seizure like discharges in neocortical brain slices. Only cells with tightly controlled resting potential revealed a narrow distribution of chloride concentration peaking at about 5 and 8 mM, in neocortical neurons and SK-N-SH cells, respectively. We thus showed that LSSmClopHensor represents a new versatile tool for studying the dynamics of chloride and proton concentration in living systems.
authors.affiliation: Institute of Biophysics, CNR , Via alla Cascata 56/C, 38123 Trento, Italy.|Bruno Kessler Foundation , Via Sommarive 18, 38123 Trento, Italy.
authors.forename: Jose M
authors.initials: JM
authors.lastname: Paredes
authors.affiliation: Institute of Biophysics, CNR , Via alla Cascata 56/C, 38123 Trento, Italy.
authors.forename: Aurora I
authors.initials: AI
authors.lastname: Idilli
authors.affiliation: Neuroscience Institute, CNR , Viale G. Colombo 3, 35121 Padova, Italy.
authors.forename: Letizia
authors.initials: L
authors.lastname: Mariotti
authors.affiliation: Neuroscience Institute, CNR , Viale G. Colombo 3, 35121 Padova, Italy.
authors.forename: Gabriele
authors.initials: G
authors.lastname: Losi
authors.affiliation: Institute of Biophysics, CNR , Via alla Cascata 56/C, 38123 Trento, Italy.|CIBIO, University of Trento , Via delle Regole 101, 38123 Trento, Italy.
authors.forename: Lyaysan R
authors.initials: LR
authors.lastname: Arslanbaeva
authors.affiliation: Nanoscience Institute, CNR , Pisa, Italy.
authors.forename: Sebastian Sulis
authors.initials: SS
authors.lastname: Sato
authors.affiliation: Scuola Normale Superiore , Pisa, Italy.
authors.forename: Pietro
authors.initials: P
authors.lastname: Artoni
authors.affiliation: Istituto Italiano di Tecnologia , Genoa, Italy.
authors.forename: Joanna
authors.initials: J
authors.lastname: Szczurkowska
authors.affiliation: Istituto Italiano di Tecnologia , Genoa, Italy.
authors.forename: Laura
authors.initials: L
authors.lastname: Cancedda
authors.affiliation: Nanoscience Institute, CNR , Pisa, Italy.|Scuola Normale Superiore , Pisa, Italy.
authors.forename: Gian Michele
authors.initials: GM
authors.lastname: Ratto
authors.affiliation: Neuroscience Institute, CNR , Viale G. Colombo 3, 35121 Padova, Italy.
authors.forename: Giorgio
authors.initials: G
authors.lastname: Carmignoto
authors.affiliation: Institute of Biophysics, CNR , Via alla Cascata 56/C, 38123 Trento, Italy.|CIBIO, University of Trento , Via delle Regole 101, 38123 Trento, Italy.
authors.forename: Daniele
authors.initials: D
authors.lastname: Arosio
mesh_terms: D000818:Animals; D015374:Biosensing Techniques / Q000379:methods*; D001923:Brain Chemistry; D002478:Cells, Cultured; D002712:Chlorides / Q000032:analysis*; D005456:Fluorescent Dyes / Q000737:chemistry*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008027:Light; D008164:Luminescent Proteins / Q000737:chemistry*; D017207:Rats, Sprague-Dawley
chemical_list: D002712:Chlorides; D005456:Fluorescent Dyes; D008164:Luminescent Proteins