Hetero-pentamerization determines mobility and conductance of Glycine receptor alpha 3 splice variants

Abstract

Glycine receptors (GlyRs) are ligand-gated pentameric chloride channels in the central nervous system. GlyR-alpha 3 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-alpha 3, 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-alpha 3 to a combined fluorescence microscopy and electrophysiology analysis. We employ masked Pearson's and dual-color spatiotemporal correlation analysis to prove that GlyR-alpha 3 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 alpha 3 heteropentamerization fine-tunes mobility and activity of GlyR-alpha 3 channels, which is important to understand and tackle alpha 3 related diseases.