Conserved High Affinity Ligand Binding and Membrane Association in the Native and Refolded Extracellular Domain of the Human Glycine Receptor α1-Subunit

The strychnine-sensitive glycine receptor (GlyR) is a ligand-gated chloride channel composed of ligand binding α- and gephyrin anchoring β-subunits. To identify the secondary and quaternary structures of extramembraneous receptor domains, the N-terminal extracellular domain (α1-(1–219)) and the large intracellular TM3–4 loop (α1-(309–392)) of the human GlyR α1-subunit were individually expressed in HEK293 cells and in Escherichia coli . The extracellular domain obtained from E. coli expression was purified in its denatured form and refolding conditions were established. Circular dichroism and Fourier-transform-infrared spectroscopy suggested ∼25% α-helix and ∼48% β-sheet for the extracellular domain, while no α-helices were detectable for the TM3–4 loop. Size exclusion chromatography and sucrose density centrifugation indicated that isolated glycine receptor domains assembled into multimers of distinct molecular weight. For the extracellular domain from E. coli , we found an apparent molecular weight compatible with a 15mer by gel filtration. The N-terminal domain from HEK293 cells, analyzed by sucrose gradient centrifugation, showed a bimodal distribution, suggesting oligomerization of ∼5 and 15 subunits. Likewise, for the intracellular domain from E. coli , a single molecular mass peak of ∼49 kDa indicated oligomerization in a defined native structure. As shown by [ 3 H]strychnine binding, expression in HEK293 cells and refolding of the isolated extracellular domain reconstituted high affinity antagonist binding. Cell fractionation, alkaline extraction experiments, and immunocytochemistry showed a tight plasma membrane association of the isolated GlyR N-terminal protein. These findings indicate that distinct functional characteristics of the full-length GlyR are retained in the isolated N-terminal domain.