The Surface Accessibility of the Glycine Receptor M2–M3 Loop Is Increased in the Channel Open State

Abstract

Mutations in the extracellular M2-M3 loop of the glycine receptor (GlyR) ␣1 subunit have been shown previously to affect channel gating. In this study, the substituted cysteine accessibility method was used to investigate whether a structural rearrangement of the M2-M3 loop accompanies GlyR activation. All residues from R271C to V277C were covalently modified by both positively charged methanethiosulfonate ethyltrimethylammonium (MTSET) and negatively charged methanethiosulfonate ethylsulfonate (MTSES), implying that these residues form an irregular surface loop. The MTSET modification rate of all residues from R271C to K276C was faster in the glycine-bound state than in the unliganded state. MTSES modification of A272C, L274C, and V277C was also faster in the glycine-bound state. These results demonstrate that the surface accessibility of the M2-M3 loop is increased as the channel transitions from the closed to the open state, implying that either the loop itself or an overlying domain moves during channel activation.

Role of Charged Residues in Coupling Ligand Binding and Channel Activation in the Extracellular Domain of the Glycine Receptor

“…We propose that conformational changes occur in the spasmodic loop and Cys-loop of the GlyR upon ligand binding that are an important part of the process linking ligand binding to channel gating. This is consistent with the previous demonstration that the spasmodic mutation results in impaired receptor gating but does not affect ligand binding (19,20), and the M2-M3 linker is involved in channel gating (12)(13)(14).…”

“…Effects of Double Charge Reversal Mutations-Charged residues located in the M2-M3 linker have previously been implicated in channel gating (12,13) and this study has demonstrated the role of loop 2 and loop 7 charged residues in gating. Therefore, we tested for electrostatic interactions between the negatively charged residues of loops 2 and 7, and the positively charged Lys-276 residue of the M2-M3 linker by creating the double reverse charge mutations E53K:K276E, D57K:K276E, and D148K:K276E.…”

“…An inherited mutation in the M2-M3 region of the nAChR is associated with a form of congenital myasthenic syndrome (10) and a mutation in the GABA A R is associated with a rare form of epilepsy (11), both of which disrupt channel gating. In addition, site-directed mutagenesis and covalent modification studies have demonstrated that the M2-M3 region undergoes a conformational change that is associated with activation of the GlyR (12,13). Similar evidence of conformational change of the M2-M3 domain has been found for the GABA A R (14).…”

“…Site-directed mutagenesis was performed using the oligonucleotidedirected polymerase chain reaction (PCR) technique and confirmed by DNA sequencing of the complete plasmid. To ensure that the sulfhydryl reagents only reacted with the substituted cysteine residues, we used receptor subunits in which the free cysteine residue at position 41 in the extracellular domain had been mutated to an alanine (13). All of the substituted cysteine mutations were introduced on this C41A background.…”

Comparison of Taurine- and Glycine-induced Conformational Changes in the M2-M3 Domain of the Glycine Receptor

“…Differences in the cysteine modification rate between the closed and open states may provide information about the associated structural changes. Our laboratory previously used this approach to demonstrate a glycine-induced increase in the surface exposure of six continuous residues (Arg 271 -Lys 276 ) in the GlyR M2-M3 linker domain (14). These residues lie mid-way between the binding site and the activation gate (7), and it is now well established that they experience a conformational change that is crucial for the activation of GlyRs (14 -18), ␥-aminobutyric acid, type A receptors (19 -24), and nAChRs (7,(25)(26)(27).…”