The structure of the TM2-3 linker in the [alpha]1 GlyR and its role in gating and modulation

The glycine receptor (GlyR) is the major inhibitory ligand-gated ion channel in the brainstem and spinal cord. It is a member of the Cys-loop superfamily of ligand-gated ion channels that includes serotonin-3, GABA[subscript A] and nicotinic acetylcholine (nAChR) receptors. Individual subunits are comprised of a large extracellular N-terminal agonist binding domain, four transmembrane (TM) segments and a large cytoplasmic loop between TM3 and TM4, containing phosphorylation sites (Brejc et al. 2001, Unwin, 2005). These receptors are pentameric in structure, with the TM2 region of each subunit contributing to the formation of a central ion pore (Lynch 2004). While the TM2-3 linker region has been hypothesized to be important for signal transduction thoughout the Cys-loop family, the precise structure and function of this region is unclear. We hypothesized that the TM2-3 linker region is a point of connection between subunits. We used disulfide bond trapping to show that the TM2-3 is able to interact with adjacent subunits and plays a critical role in signal transduction. In addition, we provide experimental evidence that the structure of the TM2-3 linker region in the [alpha]1 GlyR is a [beta]-sheet. We next sought to determine the role of the TM2-3 linker region in allosteric modulation. Using two-electrode voltage clamp electrophysiology we found that the TM2-3 linker can determine the direction of modulation without affecting modulator binding. Finally, we wanted to determine if a single alcohol and anesthetic binding site could be occupied to prevent EtOH molecules from binding. Using a combination of thiol reagents and disulfide bond trapping we show that a residue previously identified as important for the binding of alcohols and anesthetics interacts within the pore. We were unable to increase the volume at residue-267 such that EtOH was unable to bind, suggesting that EtOH may have more than one binding pocket. Together, these findings suggest that the TM2-3 linker plays a critical role in signal transduction and receptor modulation providing a foundation for future work on this region in the GlyR.