Abstract
Ion channels are involved in many physiological processes and are attractive targets for therapeutic intervention. Their functional properties vary according to their subunit composition, which in turn varies in a developmental and tissue-specific manner and as a consequence of pathophysiological events. Understanding this diversity requires functional analysis of ion channel properties in large numbers of individual cells. Functional characterisation of ligand-gated channels involves quantitating agonist and drug dose-response relationships using electrophysiological or fluorescence-based techniques. Electrophysiology is limited by low throughput and high-throughput fluorescence-based functional evaluation generally does not enable the characterization of the functional properties of each individual cell. Here we describe a fluorescence-based assay that characterizes functional channel properties at single cell resolution in high throughput mode. It is based on progressive receptor activation and iterative fluorescence imaging and delivers >100 dose-responses in a single well of a 384-well plate, using α1-3 homomeric and αβ heteromeric glycine receptor (GlyR) chloride channels as a model system. We applied this assay with transiently transfected HEK293 cells co-expressing halide-sensitive yellow fluorescent protein and different GlyR subunit combinations. Glycine EC50 values of different GlyR isoforms were highly correlated with published electrophysiological data and confirm previously reported pharmacological profiles for the GlyR inhibitors, picrotoxin, strychnine and lindane. We show that inter and intra well variability is low and that clustering of functional phenotypes permits identification of drugs with subunit-specific pharmacological profiles. As this method dramatically improves the efficiency with which ion channel populations can be characterized in the context of cellular heterogeneity, it should facilitate systems-level analysis of ion channel properties in health and disease and the discovery of therapeutics to reverse pathological alterations.
Highlights
Ion channels are involved in most physiological and disease processes [1,2,3] and are considered highly attractive drug targets for therapeutic intervention [4,5,6,7]
Our results extend the applicability of the assay to functional phenotyping of glycine receptor (GlyR) in heterogeneous cell cultures for identification of subtype-specific drugs and verify its suitability as a versatile platform for cell-based ion channel-targeted drug screening
High-resolution patch-clamp electrophysiology is still limited in throughput compared to, e.g. fluorometric approaches that allow experimentation in high-throughput mode but that are low in resolution
Summary
Ion channels are involved in most physiological and disease processes [1,2,3] and are considered highly attractive drug targets for therapeutic intervention [4,5,6,7]. Their biophysical and pharmacological properties are determined by the combination of subunits, which in the case of ligand-gated channels is often heterogeneous. The biophysical and pharmacological properties of ligand-gated ion channels are typically evaluated by analysing activation and inhibition concentration-responses and derived measures, in particular half-maximal activation or inhibition concentration (EC50, IC50), hill coefficient (nH or slope) and dynamic range of the response. While there are various technologies available that allow concentration-response experimentation with ion channels, such as flow cytometry [15,16], dynamic mass redistribution [17] or radioactive, non-radioactive and spectroscopic measurements the most commonly applied methods are patch clamp electrophysiology and fluorescence-based functional imaging [7,18,19,20]
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