The simultaneous optogenetic sensing of the intracellular concentrations of Cl− and H+ is a challenging task, which requires probes with high sensitivity allowing reliable quantitative measurements without perturbation of cell functioning. The recent paper in Frontiers of Molecular Neurosciences by Mukhtarov et al. (2013) describes the intracellular calibration and functional characterization of three genetically encoded probes developed for these purposes. The first is recently proposed combined Cl−/pH sensor (ClopHensor), which was obtained by fusion of a red fluorescent protein (RFP) with a GFP variant, E2GFP, which contains a specific Cl−-binding site (Arosio et al., 2010). The second is PalmPalm-ClopHensor, a variant that is preferentially expressed at the plasma membrane thanks to the addition of two palmitoylation sites at the N-terminus. The third ClopHensor variant contains a two additional mutations (H148G/V224L) in the GFP moiety conferring improved Cl− affinity and reduced pH dependence.
For functional analysis, constructs were expressed in CHO cells and neurons. In CHO cells ClopHensor and the H148G/V224L mutant exhibits cytoplasmic intracellular distribution while the PalmPalm-ClopHensor construct, as expected, is preferentially localized in the vicinity of membranes (FIG.1).
In order to evaluate the dynamic range and sensitivity of constructs to ions, CHO cells were co-transfected with Cl−-selective glycine receptor (GlyR) channels and one of Cl−/pH sensors. Simultaneous monitoring of fluorescent signals and ionic currents during whole-cell recordings with pipettes containing different Cl− concentrations was performed. This approach allowed to measure that ClopHensor and PalmPalm-ClopHensor bind Cl with a dissociation constant (Kd) of about 40 mM while the Kd for H148G/V224L was about 20 mM indicating its higher affinity to Cl at the physiological pH range. Compared to ClopHensor, however, the novel double mutant exhibited a smaller dynamic range, which could be a limiting factor for monitoring of small changes in Cl− concentration.
Calibration of pH, performed using the β-escin method (Waseem et al., 2010) showed that the pKa for the ClopHensor is about 6.4, while for H148G/V224L mutant it was 7.3, i.e. strongly shifted to alkaline values. In vitro analysis demonstrated that Kd for Cl− of the H148G/V224L mutant exhibits relatively small pH dependency over a wide pH range: from about 18 mM at pH 6.5 to about 30 mM at pH 7.8. This suggests that the H148G/V224L mutant is a useful tool for [Cl−]i measurements in experimental models with high pH variations.
Comparative analyses demonstrate that: (i) all three sensors can be easily expressed in various cell types and detected in different, even very small, areas of cells; (ii) all constructs exhibit remarkable stability, providing an excellent tool for long-lasting reliable monitoring and with variable acquisition rate; (iii) H148G/V224L mutant exhibits relatively small pH dependency of Cl sensitivity suggesting that this mutant can be a useful tool for [Cl−]i measurements in experimental models with high pH variations; (iv) membrane-targeted Palm-Palm-ClopHensor exhibit about 2.5-fold higher resolution of Cl transients than ClopHensor suggesting it as the preferable tool for monitoring of Cli transients on weak stimuli.
Altogether this study propose three ClopHensor constructs as promising tools for stable, long-lasting, non-invasive monitoring of [Cl−]i and pHi in different cell types. These probes are non-toxic, capable of staying in cells for a long time, can be expressed in specific cellular compartments and are suitable for production of transgenic models.
- Arosio D, Ricci F, Marchetti L, Gualdani R, Albertazzi L, Beltram F (2010) Simultaneous intracellular chloride and pH measurements using a GFP-based sensor. Nat Methods., 7(7):516-518.
- Mukhtarov, M., Liguori, L., Waseem, T., Rocca, F., Buldakova, S., Arosio, D., Bregestovski P. (2013). Calibration and functional analysis of three genetically encoded Cl-/pH sensors. Front. Mol. Neurosci. 6:9. doi: 10.3389/fnmol.2013.00009
- Waseem T., Mukhtarov M., Buldakova S., Medina I., Bregestovski P. (2010) Genetically encoded Cl-Sensor as a tool for monitoring of Cl-dependent processes in small neuronal compartments J. Neurosci.Methods, 193(1):14-23.