A State-Dependent Salt-Bridge Interaction Exists across the β/α Intersubunit Interface of the GABA_AReceptor

Abstract

The GABA A receptor is a multisubunit protein that transduces the binding of a neurotransmitter at an intersubunit interface into the opening of a central ion channel. The structural components that mediate the steps involved in this action are poorly defined. A large amount of work has focused on clarifying the specific functions and interactions of residues believed to surround the GABA binding pocket. Here, we explored two charged residues (␤ 2 Asp163 and ␣ 1 Arg120), which have been suggested by homology models to participate in a salt-bridge interaction. When mutated to alanine, both single mutants, as well as the double mutant, increase EC 50-GABA , decrease the GABA binding rate, and accelerate deactivation and GABA unbinding rates. Double-mutant cycle analysis demonstrates that the effects of each alanine mutation on the GABA binding rate were additive and independent. In contrast, a significant coupling energy was found during an analysis of deactivation time constants. Using kinetic modeling, we further demonstrated that the GABA unbinding rates, in particular, are strongly coupled. These data suggest that ␤ 2 Asp163 and ␣ 1 Arg120 form a state-dependent salt bridge, interacting when GABA is bound to the receptor but not when the receptor is in the unbound state.

Sequences Prior to Conserved Catalytic Motifs of Polysialyltransferase ST8Sia IV Are Required for Substrate Recognition

“…Arginine is favored in "hot spots" of binding energy because it is capable of multiple types of favorable interactions, including forming up to five hydrogen bonds and a salt bridge utilizing the positive charge on its guanidinium moiety (67). In fact, an arginine-aspartate salt bridge is found at the interface of the ␣-and ␤-subunits of the GABA A receptor, and removal of this bridge through alanine mutagenesis compromises GABA binding and subunit association (68). Applying these observations to the polyST-NCAM interaction, Arg 82 and Arg 93 may promote recognition through the formation of hydrogen bonds and salt bridges with residues in NCAM.…”

Interactions among Positions in the Third and Fourth Membrane-associated Domains at the Intersubunit Interface of the N-Methyl-d-aspartate Receptor Forming Sites of Alcohol Action

“…In this study we used both two-way analysis of variance and mutant cycle analysis of log-transformed ethanol IC 50 values to test for interactions among mutated positions. Although half-maximal concentration values are complex and represent multiple kinetic rates, previous studies have shown the suitability of mutant cycle analysis of complex macroscopic measures to indicate side-chain interactions (40,42,43). In addition, ethanol IC 50 values may represent fewer kinetic rates compared with agonist EC 50 values, because the main action of ethanol on NMDA receptor kinetics is to decrease mean open time (7), whereas agonist EC 50 values depend on multiple microscopic rates underlying both agonist binding and ion channel gating (44).…”

A tight coupling between β2Y97 and β2F200 of the GABAA receptor mediates GABA binding

“…According to the homology model, α 1 R120 is relatively distant from the aromatics and it has been proposed to participate in a state‐dependent inter‐subunit salt bridge with β 2 D163 (Cromer et al. 2002; Laha and Wagner 2011). The fact that α 1 R67 does not interact with the aromatics examined here but is located nearby and, on its own, severely affects GABA affinity suggest that it plays a critical role in GABA binding that is independent of the Y97/F200 pair.…”