Magnitude of a Conformational Change in the Glycine Receptor β1-β2 Loop Is Correlated with Agonist Efficacy

Abstract

The efficacy of agonists at Cys-loop ion channel receptors is determined by the rate they isomerize receptors to a pre-open flip state. Once the flip state is reached, the shut-open reaction is similar for low and high efficacy agonists. The present study sought to identify a conformational change associated with the closed-flip transition in the ␣1-glycine receptor. We employed voltage-clamp fluorometry to compare ligand-binding domain conformational changes induced by the following agonists, listed from highest to lowest affinity and efficacy: glycine > ␤-alanine > taurine. Voltage-clamp fluorometry involves labeling introduced cysteines with environmentally sensitive fluorophores and inferring structural rearrangements from ligand-induced fluorescence changes. Agonist affinity and efficacy correlated inversely with maximum fluorescence magnitudes at labeled residues in ligand-binding domain loops D and E, suggesting that large conformational changes in this region preclude efficacious gating. However, agonist affinity and efficacy correlated directly with maximum fluorescence magnitudes from a label attached to A52C in loop 2, near the transmembrane domain interface. Because glycine experiences the largest affinity increase between closed and flip states, we propose that the magnitude of this fluorescence signal is directly proportional to the agonist affinity increase. In contrast, labeled residues in loops C, F, and the pre-M1 domain yielded agonist-independent fluorescence responses. Our results support the conclusion that a closed-flip conformation change, with a magnitude proportional to the agonist affinity increase from closed to flip states, occurs in the microenvironment of Ala-52. Glycine receptors (GlyRs)3 are pentameric chloride-selective ion channels that mediate fast inhibitory neurotransmission (1). They are members of the Cys-loop receptor family that includes the prototypical nicotinic acetylcholine receptor (nAChR), the ␥-aminobutyric acid type-A receptors (GABA A Rs), and serotonin type-3 receptors (5-HT 3 Rs). Recent structural studies have provided a wealth of information on the structure and function of this receptor family (2-6). In Cys-loop receptors, the ligand-binding domain (LBD) preceding the four transmembrane helices consists of two twisted ␤-sheets. The inner (vestibule facing) ␤-sheet comprises seven ␤-strands, while the outer ␤-sheet is formed by three ␤-strands (3). The ligand binding site is located at the interface of adjacent subunits and is lined by six domains: three loops from the principal and the complementary sides, termed A-C and D-F, respectively (3).GlyRs are activated by endogenous amino acid agonists in the following order of efficacy: glycine Ͼ ␤-alanine Ͼ taurine (7, 8). As these amino acids share considerable structural similarity (Fig. 1A), they are likely to compete for the same binding site (9 -11). A recent ground-breaking study on an intermediate pre-open state, the so-called "flip" state (12), has provided new insights into the mechanism of partial agon...

A Cation-π Interaction at a Phenylalanine Residue in the Glycine Receptor Binding Site Is Conserved for Different Agonists

“…It is noteworthy, therefore, that substitutions of Phe207 in loop C had severe effects on ␤-alanine-and taurine-induced currents (taurine Ͼ ␤-alanine) but little effect on glycine-induced currents. Taken together, these results suggest that glycine, ␤-alanine, and taurine may each induce distinct conformational changes in and around the GlyR binding site (taurine Ͻ ␤-alanine Ͻ glycine), an idea that is also supported by a recent study using fluorescent reporter groups (Pless and Lynch, 2009).…”

Crystal Structures of a Cysteine-modified Mutant in Loop D of Acetylcholine-binding Protein

“…The important role of loop C derives from a large body of work using x-ray crystallography (11,(35)(36)(37), molecular dynamics simulation (38 -40), site-directed mutagenesis (41)(42)(43)(44), and electrophysiology (45,46). The data from these studies demonstrates that loop C is flexible in the non-liganded form (22,37) and adopts distinct conformations upon agonist or antagonist binding.…”

Subunit stoichiometry and arrangement in a heteromeric glutamate-gated chloride channel

“…Based on the capability of the WT homomeric GluClα (GluClαWT) receptor to respond to Glu only following exposure to IVM, it was suggested that IVM binding induces a conformational change that enables coupling of Glu binding at α/α intersubunit interfaces to the opening of the ion-channel gate (14,23). To explore this suggestion further, we used a strategy of microchimerism that is based on previous studies showing that in various Cys-loop receptors, the β1β2, Cys, and β8β9 loops of the LigBD interact with the M2-M3 loop of the pore domain to couple neurotransmitter binding to channel gating (23,(35)(36)(37)(38)(39)(40)(41)(42)(43)(44) (e.g., Fig. 1 A and C).…”

“…In various Cys-loop receptors, the β1β2, Cys, and β8β9 loops were shown to play a key role in transducing the agonist-binding energy into ion-channel gating force (35)(36)(37)(38)(39)(40)(41)(42)(43)(44). Here, we first demonstrated that although the homomeric GluClαR is not responsive to Glu, the β1β2, Cys, and β8β9 loops of the GluClα subunit are fully capable of coupling Glu binding to channel gating in a heteromeric GluClα/β microchimera that has the sequences of the α-subunit loops.…”