Pentameric ligand-gated ion channels (pLGICs) are a family of membrane proteins that mediate fast neurotransmission in the brain. The glycine receptor (GlyR) is an anion-permeable pLGIC that mediates inhibitory neurotransmission in the spinal cord, retina, and brainstem. Although the GlyR α1 subunits are widely distributed, α3-containing GlyRs are expressed predominantly in inhibitory synapses on spinal nociceptive neurons. Thus, the α3 GlyR has emerged as a therapeutic target for analgesia, and indeed, drugs that specifically enhance α3 GlyR currents are effective in treating inflammatory and neuropathic pain.
Inflammatory pain sensitization is initiated by prostaglandin E2-mediated phosphorylation of Ser346 in the α3 glycine receptor (GlyR) intracellular TM3-TM4 domain. Phosphorylation inhibits glycinergic synaptic currents, leading to disinhibition of nociceptive projection neurons and heightened pain sensation. In the first study we compared glycine-mediated conformational changes in α1 and α3 GlyRs to identify structural differences and using voltage-clamp fluorometry (VCF).We showed that glycine-mediated conformational changes in the extracellular TM2-TM3 channel gating domain were indeed significantly different between the two GlyR isoforms. Testing the hypothesis that α3 phosphorylation may also induce extracellular structural changes, we showed using both VCF and pharmacology that Ser346 phosphorylation elicits structural changes in both the TM2-TM3 loop and the α3 glycine-binding site. By demonstrating that phosphorylation alters α3 GlyR glycine-binding site structure, these results raise the possibility of developing analgesics that selectively target disease-affected GlyRs.
We previously showed that the dramatic difference in glycine efficacies of α1 and α3 GlyRs is largely attributable to their nonconserved TM4 domains. Thus, the second study investigated whether TM4 domain conformations differed between α1 and α3 GlyRs. Here we employed VCF to test whether their TM4 domains interact differently with their respective TM3 domains. We found a rhodamine fluorophore covalently attached to a homologous TM4 residue in each receptor interacts differentially with a conserved TM3 residue. We conclude that the α1 and α3 GlyRs TM4 domains are orientated differently relative to their TM3 domains. This may underlie their differential ability to influence glycine efficacy.
Hereditary hyperekplexia, or startle disease, is a neuromotor disorder caused mainly by α1 subunit mutations that either prevent the surface expression of, or modify the function of the GlyR. In the third study we demonstrate that α1β GlyR channel function is less sensitive to hyperekplexiamimicking mutations introduced into the TM2–TM3 loop of the β than into the α1 subunit. This suggests that the TM2–TM3 loop of the α1 subunit dominates the β subunit in gating α1β GlyRs.
Using VCF, we revealed that agonist-induced conformational changes in the β subunit TM2–TM3 loop were uncoupled from α1β GlyR channel gating. This is in contrast to the α subunit, where the TM2–TM3 loop conformational changes were shown to be directly coupled to α1β GlyR channel gating. Our study provides a possible explanation of why hereditary hyperekplexia-causing mutations that modify α1β GlyR channel function are almost exclusively located in the α1 to the exclusion of the β subunit. Finally, the GlyR α1 R271Q and R271L hyperekplexia mutations severely disrupt gating. We showed that when a 12-amino-acid TM2-TM3 segment incorporating the 271 hyperekplexia mutation from the β subunit was substituted for the homologous segment from the α1 subunit, that glycine sensitivity level was restored to that of the wild-type α1 GlyR. We concluded that the 271 residue is shifted out of the allosteric signalling pathway in the chimeric GlyR. We propose that this mechanism provides a novel drug design strategy not only for GlyR α1 R271Q/L-caused hereditary hyperekplexia, but also for any pathological condition that is caused by missense mutation- or covalent modification-induced disorders involving residues in allosteric signalling pathways.