A Salt Bridge Crucial for Glycine Receptor Function

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The glycine receptor (GlyR) chloride channel is a member of the pentameric ligand-gated ion channel (pLGIC) receptor family. These channels mediate fast inhibitory transmission in the nervous system. Recent structural data and a wide body of functional data have underlined the importance of the interface between the ligand-binding domain and the transmembrane domain in channel gating. A number of charged residues at this interface have been shown to form salt bridges critical for channel function in other pLGICs. However, no such interaction has been demonstrated in the GlyR. Mutations at D148 (Cys-loop) and R218 (pre-M1) in the homomeric alpha 1 GlyR can have drastic effects on GlyR function and the single mutants D148C and R218C failed to produce glycine-induced currents in Xenopus oocytes. However, the D148C, R218C double mutant resulted in receptors that produced glycine-gated currents with only a small decrease in glycine sensitivity compared to WT. Importantly, sub-saturating glycine-evoked currents were dramatically increased by the reducing agent dithiothreitol. This suggests that D148C and R218C can form a disulphide bridge, possibly indicating an electrostatic interaction between D148 and R218 that stabilizes the channel closed state. The possibilty of a state-dependent salt bridge between D148 and R218 raised the question how the charges are stabilised in the open state. Interestingly, structural studies have suggested that R218 is flanked by two highly conserved phenylalanine side chains. To test, if these aromatic amino acids interact with R218 electrostically via a cation-pi interaction, we incorporated a series of fluorinated Phe derivatives using the in vivo non-sense suppression method. However, the fluorination-induced changes in the glycine sensitivity were not consistent with a cation-pi interaction. Taken together, these results point towards a crucial salt bridge between D148 and R218 but no electrostatic contributions by the flanking phenylalanines.