Synchronous Bioimaging of Intracellular pH and Chloride Based on LSS Fluorescent Protein.

Autor:	Paredes JM; Institute of Biophysics, CNR , Via alla Cascata 56/C, 38123 Trento, Italy.; Bruno Kessler Foundation , Via Sommarive 18, 38123 Trento, Italy., Idilli AI; Institute of Biophysics, CNR , Via alla Cascata 56/C, 38123 Trento, Italy., Mariotti L; Neuroscience Institute, CNR , Viale G. Colombo 3, 35121 Padova, Italy., Losi G; Neuroscience Institute, CNR , Viale G. Colombo 3, 35121 Padova, Italy., Arslanbaeva LR; Institute of Biophysics, CNR , Via alla Cascata 56/C, 38123 Trento, Italy.; CIBIO, University of Trento , Via delle Regole 101, 38123 Trento, Italy., Sato SS; Nanoscience Institute, CNR , Pisa, Italy., Artoni P; Scuola Normale Superiore , Pisa, Italy., Szczurkowska J; Istituto Italiano di Tecnologia , Genoa, Italy., Cancedda L; Istituto Italiano di Tecnologia , Genoa, Italy., Ratto GM; Nanoscience Institute, CNR , Pisa, Italy.; Scuola Normale Superiore , Pisa, Italy., Carmignoto G; Neuroscience Institute, CNR , Viale G. Colombo 3, 35121 Padova, Italy., Arosio D; Institute of Biophysics, CNR , Via alla Cascata 56/C, 38123 Trento, Italy.; CIBIO, University of Trento , Via delle Regole 101, 38123 Trento, Italy.
Jazyk:	angličtina
Zdroj:	ACS chemical biology [ACS Chem Biol] 2016 Jun 17; Vol. 11 (6), pp. 1652-60. Date of Electronic Publication: 2016 Apr 07.
DOI:	10.1021/acschembio.6b00103
Abstrakt:	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.
Databáze:	MEDLINE
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