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

M Mukhtarov,F Rocca,D Arosio,S Buldakova,T Waseem,L Liguori,P Bregestovski

doi:10.3389/fnmol.2013.00009

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 pKa = 6.8 for H+ and Kd 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 pKa 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°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.

Highlights

Beginning with the pioneer studies by Tsien and co-authors on measurements of intracellular calcium concentration in intact lymphocytes (Tsien et al, 1982), non-invasive monitoring of different intracellular ions (Ca2+, Mg2+, Cl−, and H+) has became a powerful direction of research for functional analysis of neurons and other cell types under normal and pathophysiological conditions.Among several methods proposed for monitoring intracellular Cl− concentration ([Cl−]i), the most promising is the use of genetically encoded Cl−-sensitive probes (Bregestovski et al., 2009; Mancuso et al, 2011)
We present calibration and functional analysis of ClopHensor and two of its derivatives: (i) PalmPalm-ClopHensor, which should have preferable membrane targeting and allow near-membrane measurement of [Cl−]i and pHi; and (ii) ClopHensor with mutations H148G and V224L aimed to increase the affinity of this probe for Cl− and change the pH-sensing properties
An evident pHi increase was recorded only when there was a substantial difference between Vh and Er and relatively long (1-s) glycine application, which induced a [Cl−]i elevation of about 10 mM (Figure 9C, bottom trace). These results demonstrate that PalmPalm-ClopHensor, which has preferable membrane targeting, could be of interest for nearmembrane measurement of intracellular Cl− and pH changes

Summary

Introduction

Beginning with the pioneer studies by Tsien and co-authors on measurements of intracellular calcium concentration in intact lymphocytes (Tsien et al, 1982), non-invasive monitoring of different intracellular ions (Ca2+, Mg2+, Cl−, and H+) has became a powerful direction of research for functional analysis of neurons and other cell types under normal and pathophysiological conditions.Among several methods proposed for monitoring intracellular Cl− concentration ([Cl−]i), the most promising is the use of genetically encoded Cl−-sensitive probes (Bregestovski et al., 2009; Mancuso et al, 2011). The first generation of probes named “Clomeleon” (Kuner and Augustine, 2000) and “ClSensor” (Markova et al, 2008) was based on the Cl−-sensitive yellow-fluorescent protein (YFP) linked with Cl−-insensitive cyan-fluorescent protein (CFP), which was the reference fluorescence molecule. These indicators have opened the way for noninvasive monitoring and ratiometric measurement of [Cl−]i in different cell types in vitro (Pellegrino et al, 2011; Bertollini et al, 2012; Friedel et al, 2013). Producing a construct consisting of a glycine receptor (GlyR) with Cl-Sensor incorporated into the long cytoplasmic domain

Methods

Results

Conclusion