Synchronous Bioimaging of Intracellular pH and Chloride Based on LSS Fluorescent Protein.
- 1Institute of Biophysics, CNR , Via alla Cascata 56/C, 38123 Trento, Italy.
- 2Bruno Kessler Foundation , Via Sommarive 18, 38123 Trento, Italy.
- 3Neuroscience Institute, CNR , Viale G. Colombo 3, 35121 Padova, Italy.
- 4CIBIO, University of Trento , Via delle Regole 101, 38123 Trento, Italy.
- 5Nanoscience Institute, CNR , Pisa, Italy.
- 6Scuola Normale Superiore , Pisa, Italy.
- 7Istituto Italiano di Tecnologia , Genoa, Italy.
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.
- PMID: 27031242;
The importance of chloride
Chloride ions (Cl−) play important roles in different cellular functions and physiological processes, and its role is critical in several human diseases1, and neurodevelopmental disorders2. In particular, chloride homeostasis is increasingly investigated in neuroscience as it is involved in the synaptic signaling3, as well as in neuronal growth and migration4.
How to measure intracellular chloride
The use of non-invasive techniques in the determination of intracellular chloride brings multiple advantages, and in particular, the use of genetically encoded fluorescent biosensors is enabling us to image Cl− concentration in cells with unprecedented resolution. Cl− biosensors, however, are biased by a strong pH dependency, so that slight intracellular pH changes can lead to wrong determination of Cl− concentration. This limitation can be overcome by measuring intracellular pH and Cl- concentration simultaneously.5 In this case, the biosensor is formed by a Cl− and pH sensitivity fluorescent protein (E2GFP) and a Cl− and pH insensitivity red protein (DsRed).
Our improvement in the chloride biosensor
Despite the increasing interest in Cl− imaging, its widespread adoption is prevented by the need for a dedicated setups. To simplify these requirements and allow a wider adoption of quantitative Cl− imaging with the correct analysis of concomitant pH changes, we developed a novel biosensor exploiting a novel red fluorescent protein with a large (>100 nm) Stokes shift (i.e. a large distance between excitation and emission maxima).6 Thus in our new biosensor, named LSSmClopHensor, a single excitation source is able to excite the green and red moiety simultaneously with a considerable simplification of the required setup.
How use LSSmClopHensor
LSSClopHensor works as follows: (Figure 1)
- By using alternatively two wavelength of excitation (488 and 458 nm); we record the emission of the two different moieties of the biosensor, recording fluorescence from three channels:
o Green: Emission of E2GFP (ex 488 nm)
o Cyan: Emission of E2GFP (ex 458 nm)
o Red: Emission of LSSmKate2 (ex 458 nm)
- pH map are calculated from the ratio between Green (Cl− and pH sensitivity) and Cyan channels (Cl− sensitivity and pH insensitivity). Being both channels affected in the same way by Cl−, their ratio is determined only by the pH value. (Figure 2A) A simple calibration curve is used to derive pH values from the ratio.
- Once known the pH, we can determinate the Cl− concentration (corrected by the influence of the pH value) using the ratio of the Cyan (Cl− sensitivity and pH insensitivity) and Red channels (Cl− and pH insensitivity). (Figure 2B). A great advantage of LSSClopHensor is the precise determination of Cl− concentration even in the presence of concomitant pH changes. A previous calibration correlates the ratio with the Cl− concentration value.
In case of steady pH during measurement intervals, the use of LSSmClopHemsor is further simplified, because we only need a single wavelength of excitation (458 nm) to determine the changes in Cl− concentration.
Cl− and pH maps
We extended the use of LSSmClopHensor to monitor dynamic changes of Cl− and H+ concentration in neocortical neuron networks in ex vivo preparations. We inducted an epileptic activity to neurons and record in real time the changes in the intensity in every channel. The epileptic activity produces an alteration in the fluxes of H+ and Cl− at the plasma membrane, causing an intracellular acidification and Cl− accumulation in the imaged neurons (Figure 3).
The importance of this study
Here, we developed a new ratiometric fluorescent biosensor that requires a single excitation to optimally excite both green and red FP and exploit the ratio between their measured emissions is the used to calculate maps ofintracellular pH and Cl− concentration in time-lapse experiments.
Thus, we expect LSSmClopHensor to have wide use in imaging studies, in ex vivo and potentially also in in vivo, investigating the role of Cl− and H+ in the cellular physiology and physiopathology.
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- Piatkevich, K. D., Malashkevich, V. N., Almo, S. C., and Verkhusha, V. V. (2010) Engineering ESPT Pathways Based on Structural Analysis of LSSmKate Red Fluorescent Proteins with Large Stokes Shift. J. Am. Chem. Soc. 132, 10762−10770.
We gratefully acknowledge the financial support of Telethon Italy (Grant GGP10138), RESTATE programme cofunded by the European Union under the FP7 COFUND Marie Curie Action – Grant agreement no. 267224 to J.M.P., MIUR (PRIN2009XPTWM2) and University of Trento (Progetti di Ricerca 2014).
Daniele Arosio, Ph.D.
Institute of Biophysics
Via alla Cascata 56/C, 38123
José M. Paredes, Ph.D.
Department of Physical Chemistry
Faculty of Pharmacy
18017 Campus of Cartuja
University of Granada (Spain)