Author:

Keywords:

Science & Technology, Life Sciences & Biomedicine, Biochemistry & Molecular Biology, Cell Biology, Glycine receptors, Ligand gated ion channels, Image correlation spectroscopy, Single-molecule fluorescence, Pearson's correlation coefficient, Subunit counting, Protein co-assembly, Diffusion, Stoichiometry, Electrophysiology, Patch clamp, IMAGE CORRELATION SPECTROSCOPY, SINGLE-MOLECULE, SUBUNIT STOICHIOMETRY, MEMBRANE-PROTEINS, MAMMALIAN-CELLS, BETA-SUBUNIT, DYNAMICS, COLOCALIZATION, DIFFUSION, GEPHYRIN, Pearson’s correlation coefficient, Alternative Splicing, Ligands, Mutation, Receptors, Glycine, Synaptic Transmission, 0601 Biochemistry and Cell Biology, 0606 Physiology, 1103 Clinical Sciences, 3101 Biochemistry and cell biology, 3205 Medical biochemistry and metabolomics, 3211 Oncology and carcinogenesis

Abstract:

Glycine receptors (GlyRs) are ligand-gated pentameric chloride channels in the central nervous system. GlyR-α3 is a possible target for chronic pain treatment and temporal lobe epilepsy. Alternative splicing into K or L variants determines the subcellular fate and function of GlyR-α3, yet it remains to be shown whether its different splice variants can functionally co-assemble, and what the properties of such heteropentamers would be. Here, we subjected GlyR-α3 to a combined fluorescence microscopy and electrophysiology analysis. We employ masked Pearson's and dual-color spatiotemporal correlation analysis to prove that GlyR-α3 splice variants heteropentamerize, adopting the mobility of the K variant. Fluorescence-based single-subunit counting experiments revealed a variable and concentration ratio dependent hetero-stoichiometry. Via cell-attached single-channel electrophysiology we show that heteropentamers exhibit currents in between those of K and L variants. Our data are compatible with a model where α3 heteropentamerization fine-tunes mobility and activity of GlyR-α3 channels, which is important to understand and tackle α3 related diseases.