Final Report Summary - IMAGINGGABA (Optical real-time imaging of inhibitory GABAA receptors activity using chimeric GABA channel subunit)
• Introduction
Fluorescent proteins (FPs) have revolutionized cell biology by allowing researchers to non-invasively peer into the inner workings of cells and organisms. There is a growing interest in using fluorescent proteins to create biosensors for minimally invasive imaging of concentrations of ions and small molecules, the activity of enzymes, and changes in the conformation of proteins in living cells. Accordingly, a major effort within the field of FP research has been invested in the engineering of molecular fluorescent biosensors that are active reporters of small ions (e.g. Ca, pH, chloride, zinc), metabolites (e.g. glutamate, glucose, ATP, cAMP, lipids), signaling pathways (e.g. G protein coupled receptors, Rho GTPases), enzyme activities (e.g. protein kinase A, caspases), and reactive species (Nakai et al., 2001; Frommer et al., 2009; Tantama et al., 2012; Sample et al., 2014).
A separate but closely related branch of the biosensor development technologies deals with genetically modified ion channels. By far, only a few functional examples of such the chimeras are available. There are several reports describing Fluorescent Protein-tagged Na-K-Cl type 1 cotransporter. In these papers, authors have primarily attempted to find a functionally neutral position for tagging the pair CFP/YFP-Q69M for structural experiments in kidney cells. However, functional studies of chloride transport via this transporter were paid very little attention merely indicating a possibility for future perspectives (Pedersen et al., 2008; Monette and Forbush, 2012; Somasekharan et al., 2013). Yet another example of FP-tagged ion channels was disclosed by Zanardi et al. (2013) who developed Clophensor-ClC7. ClC-7 is a chloride-proton exchanger localized in lysosomes and in the resorption lacuna in osteoclasts where it is essential for bone resorption. Authors proposed the optical assay for monitoring the ClC-7 function in Xenopus oocytes and HEK cells. Finally, a number of FP-based probes of membrane potential have been described (Siegel and Isacoff, 1997; Sakai et al., 2001a; Ataka and Pieribone, 2002; Baker et al., 2007; Dimitrov et al., 2007; Lundby et al., 2008; Tsutsui et al., 2008).
While FP-based sensors may perform well in cell lines (i.e. HEK 293, PC12 and etc.), it has been challenging in many cases to transfer probes into neurons and still observe detectable responses (Akemann et al., 2010). All of these FP-based probes have suffered from one or more problems, including low intensity of probe fluorescence in neurons, small response magnitudes, slow kinetics of the fluorescence response and poor membrane vs intracellular localization (Perron et al., 2009).
• A summary description of the project objectives
This project has been inspired by the fact that dysfunction of the GABAergic system results in an enormous number of pathologies and an unmet need still exists for an optical tool suitable either for studying GABA channel functions in different systems or discovering their novel modulators.
The general objectives were assigned into several blocks including: (1) identification of a functionally neutral position in the mouse alpha-1-GABAAR subunit for inserting CFP/YFP-based fluorescence probe “Cl-Sensor”, and, in case of success, (2) investigation of electrophysiological and spectral properties of the resulting construct(s). Thereinafter, (3) the probe’s cellular localization was planned to be characterized by using immunostaining followed by (4) exploring its performance and functional properties in CHO cells and cultured hippocampal neurons.
• A description of the work performed since the beginning of the project
The molecule of Cl-Sensor (Markova et al., 2008) was inserted into the long cytoplasmic loop of the mouse alpha1 GABAAR subunit by random-mutagenesis techniques. The electrophysiological properties for resulting chimeric construct have been tested in comparison to those for the native subunit by whole-cell patch clamp method. Once the preservation of electrical characteristics was proved, the GABAAR-Sensor was subjected to fluorimetric analysis aimed at obtaining the spectral characteristics and calibration curves. Further, cell-type specific and cellular distribution of the GABAAR-Sensor has thoroughly been examined by immunochemistry combined with confocal imaging. Finally, we have explored the functional properties of the GABAAR-Sensor, in particular, it responsiveness to application of channel agonists, such as isoguvacine and GABA, and depolarizing stimuli, such as high K, in cultured cells and hippocampal neurons.
• A description of the main results achieved
When expressed in CHO cells or hippocampal neurons, the GABAAR-Sensor displays a preferential membrane expression profile which is very similar to that seen for control alpha-1-GFP-GABAA subunit. Comparative analysis of whole-cell recording parameters from CHO cells expressing entire GABAA receptor complex containing either the GABAAR-Sensor or alpha-1-GFP-GABA subunit have proved maintaining the receptor’s functionality in both cases. The shape of current traces, rectification and apparent EC50 were shown to be similar for the tested and control constructs indicating that insertion of the reporter into alpha-1 subunit did not affect channel characteristics.
Fluorimetric analyses have demonstrated that the GABAAR-Sensor exhibits advantageous signal to noise characteristics enabling to distinguish small alterations in chloride levels. Having determined the spectral parameters of the tested construct, we have focused our main efforts on elucidation of GABA-Sensor co-localization with other ubiquitous GABAAR subunits, beta2/beta3, as well as with the presynaptic enzyme typical for inhibitory synapses, namely GAD65/67. Using immunolabelling, GABA-Sensor was shown to be evenly distributed throughout all structural parts of hippocampal neurons. In the somatic region, co-localization with receptor clusters containing beta2/beta3 appeared to be questionable and predominantly non-specific. However, in distal dendrites primarily contributing to signal summation and output, GABA-Sensor can form uncommon but clearly evident sites of co-localization either with beta2/3 or GAD65/67 indicating that the tested construct may be tethered and enriched in inhibitory synapses.
Functional experiments in hippocampal neurons have revealed that the GABA-Sensor was able to respond to GABAAR’s agonist application. In cultured mammalian cells, application of GABA caused an increase in the intracellular chloride concentration which was blocked in the presence of antagonist of GABAAR, thereby indicating the specificity of the probe’s response.
• The final results and their potential impact and use
Genetic modification of the alpha-1-GABAAR subunit produced the functional probe preserving the most important properties, such as distribution, responsiveness, and electrochemical parameters, which are similar to those for the native subunit. Functional experiments in hippocampal neurons showed that the GABA-Sensor is sensitive to channel-specific stimulations which favourably distinguishes it from the above mentioned FP-tagged channels.
It is well known that neuronal circuits adapt in response to sensory experience, mature during development and change due to disease processes in time scales which vary from milliseconds to months. Events that extended more than a few hours in time were up to now hard or impossible to follow due to technical limitations. It is expected that pharmacologists, neurobiologists and clinicians will take advantage of the advent of the optical GABAAR-Sensor having suitable sensitivity and biocompatibility for new studies on long term physiology providing new insights into long standing questions, e.g. on how the brain couples sensory input to behavioural output, on how it fine-tunes circuitry during development, and finally also on how pathologic change and circuit dysfunction in the brain is causally manifested (Rose et al., 2014).
Fluorescent proteins (FPs) have revolutionized cell biology by allowing researchers to non-invasively peer into the inner workings of cells and organisms. There is a growing interest in using fluorescent proteins to create biosensors for minimally invasive imaging of concentrations of ions and small molecules, the activity of enzymes, and changes in the conformation of proteins in living cells. Accordingly, a major effort within the field of FP research has been invested in the engineering of molecular fluorescent biosensors that are active reporters of small ions (e.g. Ca, pH, chloride, zinc), metabolites (e.g. glutamate, glucose, ATP, cAMP, lipids), signaling pathways (e.g. G protein coupled receptors, Rho GTPases), enzyme activities (e.g. protein kinase A, caspases), and reactive species (Nakai et al., 2001; Frommer et al., 2009; Tantama et al., 2012; Sample et al., 2014).
A separate but closely related branch of the biosensor development technologies deals with genetically modified ion channels. By far, only a few functional examples of such the chimeras are available. There are several reports describing Fluorescent Protein-tagged Na-K-Cl type 1 cotransporter. In these papers, authors have primarily attempted to find a functionally neutral position for tagging the pair CFP/YFP-Q69M for structural experiments in kidney cells. However, functional studies of chloride transport via this transporter were paid very little attention merely indicating a possibility for future perspectives (Pedersen et al., 2008; Monette and Forbush, 2012; Somasekharan et al., 2013). Yet another example of FP-tagged ion channels was disclosed by Zanardi et al. (2013) who developed Clophensor-ClC7. ClC-7 is a chloride-proton exchanger localized in lysosomes and in the resorption lacuna in osteoclasts where it is essential for bone resorption. Authors proposed the optical assay for monitoring the ClC-7 function in Xenopus oocytes and HEK cells. Finally, a number of FP-based probes of membrane potential have been described (Siegel and Isacoff, 1997; Sakai et al., 2001a; Ataka and Pieribone, 2002; Baker et al., 2007; Dimitrov et al., 2007; Lundby et al., 2008; Tsutsui et al., 2008).
While FP-based sensors may perform well in cell lines (i.e. HEK 293, PC12 and etc.), it has been challenging in many cases to transfer probes into neurons and still observe detectable responses (Akemann et al., 2010). All of these FP-based probes have suffered from one or more problems, including low intensity of probe fluorescence in neurons, small response magnitudes, slow kinetics of the fluorescence response and poor membrane vs intracellular localization (Perron et al., 2009).
• A summary description of the project objectives
This project has been inspired by the fact that dysfunction of the GABAergic system results in an enormous number of pathologies and an unmet need still exists for an optical tool suitable either for studying GABA channel functions in different systems or discovering their novel modulators.
The general objectives were assigned into several blocks including: (1) identification of a functionally neutral position in the mouse alpha-1-GABAAR subunit for inserting CFP/YFP-based fluorescence probe “Cl-Sensor”, and, in case of success, (2) investigation of electrophysiological and spectral properties of the resulting construct(s). Thereinafter, (3) the probe’s cellular localization was planned to be characterized by using immunostaining followed by (4) exploring its performance and functional properties in CHO cells and cultured hippocampal neurons.
• A description of the work performed since the beginning of the project
The molecule of Cl-Sensor (Markova et al., 2008) was inserted into the long cytoplasmic loop of the mouse alpha1 GABAAR subunit by random-mutagenesis techniques. The electrophysiological properties for resulting chimeric construct have been tested in comparison to those for the native subunit by whole-cell patch clamp method. Once the preservation of electrical characteristics was proved, the GABAAR-Sensor was subjected to fluorimetric analysis aimed at obtaining the spectral characteristics and calibration curves. Further, cell-type specific and cellular distribution of the GABAAR-Sensor has thoroughly been examined by immunochemistry combined with confocal imaging. Finally, we have explored the functional properties of the GABAAR-Sensor, in particular, it responsiveness to application of channel agonists, such as isoguvacine and GABA, and depolarizing stimuli, such as high K, in cultured cells and hippocampal neurons.
• A description of the main results achieved
When expressed in CHO cells or hippocampal neurons, the GABAAR-Sensor displays a preferential membrane expression profile which is very similar to that seen for control alpha-1-GFP-GABAA subunit. Comparative analysis of whole-cell recording parameters from CHO cells expressing entire GABAA receptor complex containing either the GABAAR-Sensor or alpha-1-GFP-GABA subunit have proved maintaining the receptor’s functionality in both cases. The shape of current traces, rectification and apparent EC50 were shown to be similar for the tested and control constructs indicating that insertion of the reporter into alpha-1 subunit did not affect channel characteristics.
Fluorimetric analyses have demonstrated that the GABAAR-Sensor exhibits advantageous signal to noise characteristics enabling to distinguish small alterations in chloride levels. Having determined the spectral parameters of the tested construct, we have focused our main efforts on elucidation of GABA-Sensor co-localization with other ubiquitous GABAAR subunits, beta2/beta3, as well as with the presynaptic enzyme typical for inhibitory synapses, namely GAD65/67. Using immunolabelling, GABA-Sensor was shown to be evenly distributed throughout all structural parts of hippocampal neurons. In the somatic region, co-localization with receptor clusters containing beta2/beta3 appeared to be questionable and predominantly non-specific. However, in distal dendrites primarily contributing to signal summation and output, GABA-Sensor can form uncommon but clearly evident sites of co-localization either with beta2/3 or GAD65/67 indicating that the tested construct may be tethered and enriched in inhibitory synapses.
Functional experiments in hippocampal neurons have revealed that the GABA-Sensor was able to respond to GABAAR’s agonist application. In cultured mammalian cells, application of GABA caused an increase in the intracellular chloride concentration which was blocked in the presence of antagonist of GABAAR, thereby indicating the specificity of the probe’s response.
• The final results and their potential impact and use
Genetic modification of the alpha-1-GABAAR subunit produced the functional probe preserving the most important properties, such as distribution, responsiveness, and electrochemical parameters, which are similar to those for the native subunit. Functional experiments in hippocampal neurons showed that the GABA-Sensor is sensitive to channel-specific stimulations which favourably distinguishes it from the above mentioned FP-tagged channels.
It is well known that neuronal circuits adapt in response to sensory experience, mature during development and change due to disease processes in time scales which vary from milliseconds to months. Events that extended more than a few hours in time were up to now hard or impossible to follow due to technical limitations. It is expected that pharmacologists, neurobiologists and clinicians will take advantage of the advent of the optical GABAAR-Sensor having suitable sensitivity and biocompatibility for new studies on long term physiology providing new insights into long standing questions, e.g. on how the brain couples sensory input to behavioural output, on how it fine-tunes circuitry during development, and finally also on how pathologic change and circuit dysfunction in the brain is causally manifested (Rose et al., 2014).