Genetically encoded optical sensors for monitoring of intracellular chloride and chloride-selective channel activity

Abstract

This review briefly discusses the main approaches for monitoring chloride (Cl(-)), the most abundant physiological anion. Noninvasive monitoring of intracellular Cl(-) ([Cl(-)]i) is a challenging task owing to two main difficulties: (i) the low transmembrane ratio for Cl(-), approximately 10:1; and (ii) the small driving force for Cl(-), as the Cl(-) reversal potential (E(Cl)) is usually close to the resting potential of the cells. Thus, for reliable monitoring of intracellular Cl(-), one has to use highly sensitive probes. From several methods for intracellular Cl(-) analysis, genetically encoded chloride indicators represent the most promising tools. Recent achievements in the development of genetically encoded chloride probes are based on the fact that yellow fluorescent protein (YFP) exhibits Cl(-)-sensitivity. YFP-based probes have been successfully used for quantitative analysis of Cl(-) transport in different cells and for high-throughput screening of modulators of Cl(-)-selective channels. Development of a ratiometric genetically encoded probe, Clomeleon, has provided a tool for noninvasive estimation of intracellular Cl(-) concentrations. While the sensitivity of this protein to Cl(-) is low (EC(50) about 160 mM), it has been successfully used for monitoring intracellular Cl(-) in different cell types. Recently a CFP-YFP-based probe with a relatively high sensitivity to Cl(-) (EC(50) about 30 mM) has been developed. This construct, termed Cl-Sensor, allows ratiometric monitoring using the fluorescence excitation ratio. Of particular interest are genetically encoded probes for monitoring of ion channel distribution and activity. A new molecular probe has been constructed by introducing into the cytoplasmic domain of the Cl(-)-selective glycine receptor (GlyR) channel the CFP-YFP-based Cl-Sensor. This construct, termed BioSensor-GlyR, has been successfully expressed in cell lines. The new genetically encoded chloride probes offer means of screening pharmacological agents, analysis of Cl(-) homeostasis and functions of Cl(-)-selective channels under different physiological and pathological conditions.

Keywords: FRET; fluorescent dyes; fluorescent proteins; glycine receptor channel; ion-sensitive microelectrodes; noninvasive monitoring; quinolinium indicators.

Figure 1

Cl⁻-sensitive microelectrodes and intracellular chloride concentration measurements. (A) Construction of recessed-tip Cl⁻-sensitive microelectrode. The complete electrode (top diagram) and enlarged view of the sensitive tip (bottom diagram) are shown. (From Neild and Thomas, 1973). (B) Diagram showing the basic experimental arrangement for chloride concentration measurements using microelectrodes. (Modified from Thomas, 1977). (C) Example of simultaneous recording of membrane potential (E_m) and [Cl⁻]_i in smooth muscle cell of the guinea pig vas deferens using double-barrelled microelectrode. Cl⁻-free and normal solutions were applied. Note the very slow (min) recorded Cl⁻ transients. (Modified from Aickin and Brading, 1982).

Figure 2

Chemical structure and fluorescent properties of widely used Cl⁻-sensitive dyes. (A) Chemical structure of quinolinium Cl⁻-sensitive dyes. Left: Common structure, R₁ and R₂, radicals. Middle and right: Structural formula for SPQ and MQAE dyes respectively. (B) Examples of continuous fluorescence measurements of the MQAE-loaded cells. Note the rapid degradation in baseline fluorescence value (top and bottom traces) and dramatic decrease in the fluorescent response for the second transition from 105 to 0 mM of Cl⁻ (top trace). (Modified from Nakamura et al., 1997). (C) Two-photon imaging in MQAE-loaded cerebellar neurons. Changes in fluorescence (top traces) and corresponding transmembrane currents (bottom traces) caused by 150-ms-long pressure applications of GABA. Note about 50-times slower fluorescent transients in comparison with currents. (Modified from Marandi et al., 2002).

Figure 3

Structure and fluorescence properties of Clomeleon. (A) Scheme of Clomeleon construct and (B) schematic reconstruction of Clomeleon structure. The fluorescent domains, CFP (cyan) and Topaz (yellow) were set in parallel orientation as N- and C-terminal ends of either domain were on the same site. The distance between CFP and Topaz chromophores was calculated to be 3.3 nm. (From Kuner and Augustine, (top) and Jose et al., (bottom). (C) Emission spectra of Clomeleon in the presence of different Cl⁻ concentrations. In all cases, the recombinant protein was excited at 434 nm and the emission spectra were normalized to their peaks at 527 nm. (D) The relationship between fluorescence emission ratio (527 nm/485 nm) and Cl⁻ concentrations. (Data from Kuner and Augustine, 2000).

Figure 4

Design and fluorescence properties of Cl-Sensor. (A) Schematic representation of Cl-Sensor construct; *** indicates three mutations: YFP-H148Q, -I152L and -V163S in the YFP sequence. (B) Normalized spectra of Cl-Sensor. Whole-cell recordings from CHO cells with pipettes containing different Cl⁻ concentrations (shown in the graph). Note that spectra have a common point at 465 nm. (C) Comparison of Cl⁻-sensitivities of Cl-Sensor and Clomeleon. Cl-Sensor (black squares and line): the relationship between fluorescence excitation ratio (F₄₈₀/F₄₄₀) and [Cl⁻]_i obtained from whole-cell recordings with pipettes containing solutions with different Cl⁻ concentrations (from 0 to 150 mM) (from Markova et al., 2008). Clomeleon (blue circles and line): the relationship between fluorescence emission ratio (F₅₂₇/F₄₈₅) and [Cl⁻]_i plotted from Kuner and Augustine, (see Figure 4D). Note that main part of calibration curve for Clomeleon is out of physiological range of [Cl⁻]_i.

Figure 5

Structure and fluorescence properties of BioSensor-GlyR. (A) Scheme of BioSensor-GlyR construct. Two subunits are shown. (B) Whole-cell currents induced by rapid application of glycine (30 or 300 μM) and dose-response curves obtained from CHO cells transfected with either wild-type human GlyR (blue) or BioSensor-GlyR (green). Note similar kinetics and agonist sensitivity for wild-type GlyR and BioSensor-GlyR. (C) Examples of simultaneous whole-cell and fluorescence recordings from BioSensor-GlyR transfected BHK cells with pipette containing either nominally 0 (left traces) or 150 mM (right traces) Cl⁻. Glycine (1 mM) was pressure applied for 10 ms duration. (D) Relationship between the amplitude of glycine-induced currents and changes in fluorescence of BioSensor-GlyR at 480 nm. The amplitude of currents was regulated by the changing of V_h. (Modified from Mukhtarov et al., 2008).