Comparative Surface Accessibility of a Pore-lining Threonine Residue (T6′) in the Glycine and GABAA Receptors

Abstract

The substituted cysteine accessibility method was used to probe the surface exposure of a pore-lining threonine residue (T6) common to both the glycine receptor (GlyR) and ␥-aminobutyric acid, type A receptor (GABA A R) chloride channels. This residue lies close to the channel activation gate, the ionic selectivity filter, and the main pore blocker binding site. Despite their high amino acid sequence homologies and common role in conducting chloride ions, recent studies have suggested that the GlyRs and GABA A Rs have divergent open state pore structures at the 6 position. When both the human ␣1 T6C homomeric GlyR and the rat ␣1 T6C ␤1 T6C heteromeric GABA A R were expressed in human embryonic kidney 293 cells, their 6 residue surface accessibilities differed significantly in the closed state. However, when a soluble cysteine-modifying compound was applied in the presence of saturating agonist concentrations, both receptors were locked into the open state. This action was not induced by oxidizing agents in either receptor. These results provide evidence for a conserved pore opening mechanism in anion-selective members of the ligand-gated ion channel family. The results also indicate that the GABA A R pore structure at the 6 level may vary between different expression systems. The ligand-gated ion channel (LGIC)1 superfamily includes the nicotinic acetylcholine receptor (nAChR), serotonin type 3 receptor (5HT 3 R), GABA A receptor (GABA A R), and glycine receptor (GlyR), as well as invertebrate glutamate and histidine receptors (1). Functional receptors of this family comprise five homologous subunits arranged in a ring to form a central ion-conducting pore. Each subunit is composed of a large extracellular ligand-binding N-terminal domain, four membranespanning segments (M1-M4), and a large intracellular domain between M3 and M4.The pore-lining, second transmembrane (M2) domain has an ␣-helical secondary structure that undergoes a conformational change as the channel is opened (2). To investigate this process in detail, state-dependent differences in the surface exposure of M2 domain residues can be assayed using the substituted cysteine accessibility method (3). In this technique, residues are mutated individually to cysteines, and changes in their reactivity rates with soluble cysteine-reactive reagents can identify structural changes between different functional states. As expected for receptors belonging to the same family, this technique has generally yielded a good correlation between the open state M2 domain secondary structures of the nAChR (4 -7), GABA A R (8), and 5HT 3 R (9, 10).The M2 domain 6Ј residue, which is a threonine in the GlyR ␣1 subunit and the GABA A R ␣1 and ␤1 subunits (see Fig. 1A), lines a critical part of the pore. It is close to the activation gate (6,11,12) and the ionic selectivity filter (13-15) and forms the main pore blocker binding site (reviewed in Ref. 16). Therefore, structural differences at this level may be expected to have significant functional consequences. In the homo...

Closed-state Cross-linking of Adjacent β1 Subunits in α1β1 GABAa Receptors via Introduced 6′ Cysteines

“…Although 100:400 M Cu:phen applied in the closed or open states had no effect on current magnitude, a 2-min application of 10 mM DTT in the closed state caused a weak transient potentiation that is most likely a pharmacological action (13). In contrast, the ␣ T6ЈC ␤…”

“…The total rotational movement, which was proposed to be around 120 o , is not easily reconciled with other studies. Specifically, Horenstein et al (12) In apparent contrast, an electrophysiological analysis of the effects of oxidizing and reducing agents on the same GABA A R subunits recombinantly expressed in HEK293 cells suggested that 6Ј cysteines efficiently formed disulfide bonds in the closed state and in the desensitized state (13). This suggests that the functional properties of identical ␣ T6ЈC ␤ T6ЈC GABA A Rs vary dramatically between the Xenopus oocyte and the HEK293 cell expression systems.…”

Minimal Structural Rearrangement of the Cytoplasmic Pore during Activation of the 5-HT3A Receptor

“…8). Although the precise location of the gate has been equivocal (8,13), the 4-Å density map of the nAChR (6) and recent MTS studies with glycine, ␥-aminobutyric acid, type A, and 5-HT 3 receptors implicate movement between the 6Ј and 13Ј positions (7,9,10,57). In support of this, linear free energy relationship analysis of mutant nAChRs suggested that the cytoplasmic side of M2 moves last upon activation after a wave of energy moving from the site of ligand binding down toward the cytoplasmic selectivity filter (61,62).…”

Structure and Function of the Glycine Receptor and Related Nicotinicoid Receptors

“…In the GlyR, residues in the M2-M3 loop, as well as the M1-M2 loop, were shown to be involved in gating (46). In addition, changes in the accessibility of residues the M2-M3 linker in GlyR and GABA A R as a function of the activation state of the receptor suggests a movement of M2-M3 loop in gating (44,47). Specific residues in the ligand-binding domain that interact with distinct residues on the M2-M3 loop of GABA A R, coupling agonist binding to channel gating, have been identified, and the interaction of the ligand-binding domain with the transmembrane domains has been elegantly modeled (48).…”

“…In addition, differential accessibilities to labeling reagents in SCAM studies have illustrated many of the structural changes in the resting, open, and desensitized state of the receptor (15,44). Photolabeling studies of the nAChR also showed differential accessibility of residues in the ␣ 1 subunit in open, closed, and desensitized states, particularly in the signature Cys-loop (45).…”