Glycine receptor α3K governs mobility and conductance of L/K splice variant heteropentamers

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 single-channel on-cell patch clamp we show heteropentameric conductances resemble those of the α3K splice variant. 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. Summary The glycine receptor α3 is key to the central nervous system’s physiology and involved in chronic pain and epilepsy. In this paper, Lemmens et al. reveal and functionally characterize α3 splice variant heteropentamerization via advanced single-molecule fluorescence image analysis. Declarations Funding We acknowledge the UHasselt Advanced Optical Microscopy Centre (AOMC). Prof. Em. Marcel Ameloot, the Research Foundation Flanders (FWO, project G0H3716N) and the province of Limburg (Belgium) (tUL Impuls II) are acknowledged for funding the microscopy hardware. V. Lemmens is grateful for a doctoral scholarship from the UHasselt (17DOC11BOF) and KU Leuven (C14/16/053) Special Research Funds (BOF). Conflicts of interest / competing interests No conflicts of interest apply. Ethics approval Not applicable Availability of data and material All data and material are available upon request. Code availability Fluctuation imaging and co-localization analyses were performed in the software package PAM [71]. The software is available as source code, requiring MATLAB to run, or as pre-compiled standalone distributions for Windows or MacOS at http://www.cup.uni-muenchen.de/pc/lamb/software/pam.html or hosted in Git repositories under http://www.gitlab.com/PAM-PIE/PAM and http://www.gitlab.com/PAM-PIE/PAMcompiled. A detailed user manual is available at http://pam.readthedocs.io. Author contributions Conceptualization Meier J.C., Brône B. and Hendrix J.; Investigation and formal analysis Lemmens V. and Thevelein B.; Software development Hendrix J.; Writing the original draft Lemmens V., Thevelein B and Hendrix, J.; Review and editing by all authors.