Calibration and functional analysis of three genetically encoded Cl-/pH sensors

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 at al., 2008; VVaseem et al., 2010). Recently, a combined Cl-/pH sensor (ClopHensor) opened the way for simultaneous ratiometric measurement of these two ions (Arosio at al., 2010). ClopHensor was obtained by fusion of a red fluorescent protein (DsRed-monomer) to the E(2)GFP 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 (similar to 73). 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 Palm Palm-ClopHensor and the H148GN224L mutant with improved Cl- affinity, reduced pH dependence, and pK, 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. Kd values for Cl measured at 33 degrees C and pH similar to 7.3 were, respectively, 39, 47 and 21 mM for ClopHensor, PaImPalm-ClopHensor, and the H148GN224L mutant. PaImPalm-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-] in various living cells.