AUTHOR=Mukhtarov Marat , Liguori Lavinia , Waseem Tanya , Rocca Francesco , Buldakova Svetlana , Arosio Daniele , Bregestovski Piotr TITLE=Calibration and functional analysis of three genetically encoded Cl−/pH sensors JOURNAL=Frontiers in Molecular Neuroscience VOLUME=6 YEAR=2013 URL=https://www.frontiersin.org/journals/molecular-neuroscience/articles/10.3389/fnmol.2013.00009 DOI=10.3389/fnmol.2013.00009 ISSN=1662-5099 ABSTRACT=

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 E²GFP 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.