Investigating structure and function in the glycine receptor chloride channel

The glycine-receptor chloride-channel (GlyR) mediates inhibitory neuro-transmission in the central nervous system. GlyR is a member of the ligand-gated ionchannel (LGIC) superfamily which have a pentameric structure comprising five subunits arranged around a central ion-channel. Each subunit comprises a large extracellular domain and four transmembrane domains (M1-M4). The M2 domain forms an a-helix that lines the channel pore. The channel activation is mediated by long-range allosteric mechanisms. The broad aim of this thesis has been to improve our understanding of the transduction pathway between the ligand-binding site and the activation gate. The b-amino acid, taurine, is a full agonist of the human glycine receptor a1 subunit when recombinantly expressed in a mammalian (HEK293) cell line, but a partial agonist of the same receptor when expressed in Xenopus oocytes. Several residues in the Ala101 n Thr112 domain have previously been identified as determinants of b-amino acid binding and gating mechanisms in Xenopus oocyte-expressed receptors. Firstly, the effect of cysteine mutagenesis of all residues in Ala101-Thr112 domain on the glycine and taurine's apparent affinity and their relative agonist efficacy were determined when the mutant GlyRs were recombinantly expressed in HEK cells. Then, the surface accessibility of each introduced cysteine was assayed by comparing the reaction rates of both positvely- and negatively-charged methanethiosulfonate (MTS) reagents in the closed-state, in the glycine- and in the taurine-bound states. The results showed that Asn102 and Glu103 are identified as taurine and glycine binding. The N102C mutation also abolished the antagonistic actions of taurine, indicating that this site does not discriminate between the putative agonist- and antagonist-bound conformations of b- amino acids. The effects of mutations from Lys104 n Thr112 indicate that the mechanism by which this domain controls b-amino acid-specific binding and gating processes differs substantially depending on whether the receptor is expressed in mammalian cells or Xenopus oocytes. Thr112 is the only domain element in mammalian cell-expressed GlyRs which was demonstrated to discriminate between glycine and taurine. In conclusion, it was proposed that this domain actually forms part of the b-amino acid antagonist allosteric transduction pathway and that the mutations which disrupt this site act by improving the allosteric coupling efficiency between the binding site and the activation gate. It has been proposed that the M2-M3 loops act as a gating control element in alanine-substituted mutations of M2-M3 loop. The peak current, agonist EC50 values and I-V relationship of cysteine-substituted mutations of this loop were investigated. The results revealed that the cysteine-substituted mutants are generally no difference to those of the corresponding alanine-substituted mutants. Eight of 12 cysteine mutations (R271C-A272C, L274C-Y279C) dramatically decrease the glycine affinity and convert taurine from full-agonist into partial- or complete-antagonists. The I-V relationships of all 12 mutants (S270C-K281C) are same as WT, indicating the M2-M3 loops may not play a role in controlling ion permeation. Since that N-terminal half of the M2-M3 loop (Arg271 -Val 277) is exposed to the aqueous environment' and this domain undergoes movement as the channel transition from the closed- to open-state, the MTS modification rates in both glycine-bound and taurine-bound states were compared to test whether glycine and taurine induce different conformational changes to this loop. The applications of MTS switch taurine from partial- or complete-antagonist back to full-agonist on A272C, L274C and increase the taurine relative peak current relative to glycine on A272C-L274C, consisting with the increase of glycine affinity. The MTS modification rates on saturating concentration of taurine are same as low concentration of glycine (EC5-EC10) on A272C-L274C indicating taurine can only induce the same conformational changes as low concentration of glycine. The modifications of MTS on the R271C and K276C are glycine dose-dependent. In low concentration of glycine that is equivalent to saturating concentration of taurine, R271C can not be accessible by MTS. Although K276C can be accessible by MTS but there is not any functional change. The result suggested that the reason of M2- M3 loop mutants converting taurine from full-agonist to partial- or complete-antagonist is that the mutations in this loop interrupt the conformational information transfer from ligand-binding site to channel gating. In summary, these studies has provided essential insight into the understanding the molecular architecture of GlyR. Since GlyR has been established as an elegant model for the knowledge of the structure and function of LGIC family, the results of this thesis will have general applicability to the insight of the structural basis of channel activation in all members of this family.n