Abstract
The strychnine-sensitive glycine receptor (GlyR) is a ligand-gated ion channel that mediates fast synaptic inhibition in the vertebrate central nervous system. As a member of the family of Cys-loop receptors, it assembles from five homologous subunits (GlyRalpha1-4 and -beta). Each subunit contains an extracellular ligand binding domain, four transmembrane domains (TM), and an intracellular domain, formed by the loop connecting TM3 and TM4 (TM3-4 loop). The TM3-4 loops of the subunits GlyRalpha1 and -alpha3 harbor a conserved basic motif, which is part of a potential nuclear localization signal. When tested for functionality by live cell imaging of green fluorescent protein and beta-galactosidase-tagged domain constructs, the TM3-4 loops of GlyRalpha1 and -alpha3, but not of GlyRalpha2 and -beta, exhibited nuclear sorting activity. Subunit specificity may be attributed to slight amino acid alterations in the basic motif. In yeast two-hybrid screening and GST pulldown assays, karyopherin alpha3 and alpha4 were found to interact with the TM3-4 loop, providing a molecular mechanism for the observed intracellular trafficking. These results indicate that the multifunctional basic motif of the TM3-4 loop is capable of mediating a karyopherin-dependent intracellular sorting of full-length GlyRs.
Highlights
Mon pentameric rosette-like composition of homologous subunits and a characteristic subunit topology (Fig. 1, A and B)
The intracellular domain of the receptor is mainly formed by the TM3– 4 loop; its protein conformation has not been resolved
We report the presence of a multifunctional basic motif that is part of a potential nuclear localization signal (NLS) within the TM3– 4 loop of adult, synaptic glycine receptor (GlyR)␣ subunits, i.e. GlyR␣1 and -␣3
Summary
Sequence Analysis and Cloning—For multiple sequence alignment, the sequences and boundaries of the TM3– 4 loops were taken from the annotation in the Uniprot data base [15]. Immunocytochemistry, GFP Imaging, and Electrophysiology— For immunocytochemistry, HEK293 cells or neurons were grown on polylysine-coated coverslips, transfected if necessary, fixed with 3% paraformaldehyde in phosphate-buffered saline (PBS), and permeabilized as well as blocked with 0.1% Triton X-100, 5% donkey or sheep serum (Dianova) in PBS for 30 min at room temperature. Cells were incubated with primary antibodies for 1 h at room temperature and washed 3 times with PBS. This procedure was repeated with secondary antibodies. Transfected neurons grown on polylysine-coated coverslips were fixed using 5% acetate in methanol for 10 min at Ϫ20 °C, embedded in Mowiol, and subjected to either confocal or standard fluorescence microscopy, the latter with an Axioskop microscope (Carl Zeiss, Jena, Germany). Chemiluminescence substrates were ECLϩ (GE Healthcare) and SuperSignal West Femto (Thermo Fischer Scientific, Waltham, MA) for normal and weak signals, respectively
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