The inhibitory glycine receptor : ligand action, molecular structure and channel gating mechanism

Shan, Qiang. (2003). The inhibitory glycine receptor : ligand action, molecular structure and channel gating mechanism PhD Thesis, School of Biomedical Sciences, The University of Queensland.

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Author Shan, Qiang.
Thesis Title The inhibitory glycine receptor : ligand action, molecular structure and channel gating mechanism
School, Centre or Institute School of Biomedical Sciences
Institution The University of Queensland
Publication date 2003
Thesis type PhD Thesis
Supervisor Dr. Joe Lynch
Total pages 210
Collection year 2003
Language eng
Subjects L
320502 Basic Pharmacology
730104 Nervous system and disorders
Formatted abstract The glycine receptor (GlyR) chloride channel is responsible for inhibitory neurotransmission in the vertebrate central nervous system. It is a member of the ligandgated ion channel (LGIC) superfamily that also includes the nicotinic acetylchoUne receptor (nAChR) and the GABAA receptor (GABAAR). This thesis sought to investigate the molecular basis of GlyR activation using a combination of site-directed mutagenesis and patch-clamp electrophysiological analysis. 

Much of this project focused on the role of a threonine residue (T6'), so numbered as it lies 6 residues in from the internal end of the pore-lining second transmembrane (M2) domain. This residue is involved in several ion channel functions including gating, ion selectivity and blocker binding.

Former work has reported that the heteromeric αβ GlyR is less sensitive to picrotoxin inhibition than the homomeric α GlyR and that this difference arises from structural differences in the pore-lining M2 segment. We investigated which residues determined this picrotoxin sensitivity difference. By analogy with structurally-related receptors, the β subunit M2 domain residues β2' and F6' were considered the most likely candidates for mediating this effect. My mutating the 6' and 2' residues in both subunits, I demonstrated that a ring of 5 threonines at the M2 domain depth corresponding to the a subunit T6' is specifically required for picrotoxin sensitivity.

The substituted cysteine accessibility method (SCAM) involves assaying the reactivity of introduced cysteines with highly soluble cysteine-specific reagents. If the reagents produce an irreversible change in some functional property, indicative of a covalent reaction, then the residue is assumed to lie at the protein aqueous interface. This approach revealed that the T6'C lines the aqueous channel pore in the open state, suggesting that picrotoxin binds to this site. The same approach revealed that T6'C was not exposed to the external aqueous environment in the closed state. This implies either that the 6' cysteine moves from the protein interior towards the channel pore in the open state or that an externally-located pore constriction precludes the access to T6'C.

However, the corresponding residues in the GABAAR expressed in Xenopus oocytes were shown by others to respond differently to cysteine-reactive reagents, suggesting an entirely different structure in this region. This surprised us as the two receptors have very similar structures and a common role in conducting chloride ions. To probe the structural basis for this difference, we expressed the same GABAAR in HEK cells. A SCAM analysis revealed the receptors had different closed state structures at the 6' region of the pore although their open state structures bore strong similarities. These results provided evidence for a conserved pore opening mechanism in anion-selective members of the ligand-gated ion channel superfamily and also indicated that the GABAAR pore structure at the 6' level may depend upon the expression system.

Finally, we used T6'C reactivity as an index of individual subunit activation to characterize the channel allosteric activation mechanism. We concluded that 6' cysteines moved only when the same subunit had been bound with an agonist molecule, which supported an uncoupled rather than a concerted model for GlyR activation. Together, the series of studies that focused on the structure and function of T6' provide important new insights into the molecular structural basis of GlyR activation.

The final project was not directly related to the above series of studies. It involved investigating the effects of the antihelminthic, ivermectin, on the GlyR. Using a variety of site-directed mutagenesis approaches, we concluded that ivermectin activates the GlyR by binding to a site distinct from the glycine site. These findings establish ivermectin as a useful new tool for dissecting GlyR molecular structure and function. 
Keyword Glycine -- Physiological effect

Document type: Thesis
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Created: Fri, 24 Aug 2007, 18:19:31 EST