Linking of Glycine Receptor Transmembrane Segments Three and Four Allows Assignment of Intrasubunit-Facing Residues

Abstract

Glycine receptors (GlyRs) are pentameric ligand-gated ion channels that mediate inhibitory neurotransmission in the brain and spinal cord and are targets of alcohols and anesthetics. The transmembrane (TM) domain of GlyR subunits is composed of four α-helical segments (TM1–4), but there are conflicting data about the orientation of TM3 and TM4 and, therefore, also the proximity of residues (e.g., A288) that are important for alcohol and anesthetic effects. In the present study, we investigated the proximity of A288 in TM3 to residues in TM4 from M404 to K411. We generated eight double mutant GlyRs (A288C/M404C, A288C/F405C, A288C/Y406C, A288C/W407C, A288C/I408C, A288C/I409C, A288C/Y410C, and A288C/K411C), as well as the corresponding single mutants, and expressed them in Xenopus laevis oocytes. To measure glycine responses, we used two-electrode voltage clamp electrophysiology. We built homology models of the GlyR using structures of the nicotinic acetylcholine receptor (nAChR) and a prokaryotic ion channel (Gloeobacter violaceus, GLIC) as templates, and asked which model best fit our experimental data. Application of the cross-linking reagent HgCl2 in the closed state produced a leftward shift in the glycine concentration–response curves of the A288C/W407C and A288C/Y410C mutants, suggesting they are able to form cross-links. In addition, when HgCl2 was coapplied with glycine, responses were changed in the A288C/Y406C, A288C/I409C, and A288C/Y410C double mutants, suggesting that agonist-induced rotation of TM4 allows A288C/Y406C and A288C/I409C to cross-link. These results are consistent with a model of GlyR, based on nAChR, in which A288, Y406, W407, I409, and Y410 face into a four-helical bundle.

A Novel Alcohol-Sensitive Position in the N-Methyl-d-Aspartate Receptor GluN2A Subunit M3 Domain Regulates Agonist Affinity and Ion Channel Gating

“…Studies designed to identify the NMDA receptor region containing the site of alcohol action found that the NMDA receptor C-terminal domain regulates ethanol sensitivity (Alvestad et al, 2003;Xu et al, 2011;Trepanier et al, 2012); however, this domain does not contain the primary site of alcohol action because truncation of the C-terminal domain does not abolish ethanol inhibition (Anders et al, 2000;Peoples and Stewart, 2000). Taken together with studies demonstrating the role of membraneassociated domains in NMDA receptor gating (Kohda et al, 2000;Jones et al, 2002;Sobolevsky et al, 2002a), these studies suggested that sites of ethanol action may be located in one or more of the membrane-associated (M) domains, as appears to be the case for alcohol and anesthetic sites on GABA A and glycine receptors (Mihic et al, 1997;Lobo et al, 2008;McCracken et al, 2010).…”

Structure and Pharmacologic Modulation of Inhibitory Glycine Receptors

“…2D) comprises the S267 (TM2) and A288 residues (TM3) complemented by Q266 and M287. Besides this site, additional binding pockets for ethanol have been proposed in the TM4 domain (i.e., I409, Y410, and K411) (Lobo et al, 2006;McCracken et al, 2010a) and in the ECD (i.e., A52) (Crawford et al, 2007;Perkins et al, 2008). Thus, it is likely that several binding pockets for ethanol exist on GlyR, and they should become occupied as the concentration of the ligand increases.…”

Characterization of Two Mutations, M287L and Q266I, in the α1 Glycine Receptor Subunit That Modify Sensitivity to Alcohols

“…The modeling was similar to that described previously (McCracken et al, 2010a). Although the possibilities for alignment of the TM3 segment of GlyR with GABA A and nicotinic acetylcholine receptors have been a subject of some controversy (Ernst et al, 2005), we chose the "no gap" alignment of GlyR with GLIC as suggested by Bocquet et al (2009).…”

“…Because alcohol binding cannot be studied by traditional radiolabel binding, mutating critical amino acids to cysteine and irreversibly labeling them with thiol-specific alcohol analogs allowed the determination of specific amino acids involved in alcohol binding and action (Mascia et al, 2000). Subsequent research expanded these findings and identified critical amino acids in TM1 and TM4 (Lobo et al, 2008;McCracken et al, 2010a).…”