Structural model of ρ1 GABA_C receptor based on evolutionary analysis: Testing of predicted protein–protein interactions involved in receptor assembly and function

Abstract

The homopentameric q1 GABA C receptor is a ligand-gated ion channel with a binding pocket for c-aminobutyric acid (GABA) at the interfaces of N-terminal extracellular domains. We combined evolutionary analysis, structural modeling, and experimental testing to study determinants of GABA C receptor assembly and channel gating. We estimated the posterior probability of selection pressure at amino acid residue sites measured as x-values and built a comparative structural model, which identified several polar residues under strong selection pressure at the subunit interfaces that may form intersubunit hydrogen bonds or salt bridges. At three selected sites (R111, T151, and E55), mutations disrupting intersubunit interactions had strong effects on receptor folding, assembly, and function. We next examined the role of a predicted intersubunit salt bridge for residue pair R158-D204. The mutant R158D, where the positively charged residue is replaced by a negatively charged aspartate, yielded a partially degraded receptor and lacked membrane surface expression. The membrane surface expression was rescued by the double mutant R158D-D204R, where positive and negative charges are switched, although the mutant receptor was inactive. The single mutants R158A, D204R, and D204A exhibited diminished activities and altered kinetic profiles with fast recovery kinetics, suggesting that R158-D204 salt bridge perhaps stabilizes the open state of the GABA C receptor. Our results emphasize the functional importance of highly conserved polar residues at the proteinprotein interfaces in GABA C q1 receptors and demonstrate how the integration of computational and experimental approaches can aid discovery of functionally important interactions.

Differentiating Enantioselective Actions of GABOB: A Possible Role for Threonine 244 in the Binding Site of GABAC ρ1 Receptors

“…45 The amino acid sequence of GABA C ρ 1 receptor (accession code: P24046) as obtained from NCBI 46 was aligned on the template. Sequence alignments were based on the results of Adamian et al, 47 …”

Structure-function study of the fourth transmembrane segment of the GABAρ1 receptor

“…Nevertheless, homology-based computer modeling provides sufficiently accurate structural details to test the potential role of functional domains of the receptor (12)(13)(14)(15). We are interested in understanding the functional role of the carboxy-terminus of the TM4 segments of GABAρ receptors.…”

GABA Receptors: Pharmacological Potential and Pitfalls

“…Furthermore, several key structural elements that determine specific pharmacological response of GABAC receptors have been found [228]. Namely, mutational studies, including those directed toward N-terminal domain and transmembrane domain TM4, have revealed residues that change sensitivity to agonists or make GABAC complex inactive, and contribute to the binding pocket determining properties of GABA binding [221,230,231]. Thus, Tyr102 at ρ1 subunit was identified as part of GABA binding domain, and probably the important residue for coupling agonist binding to channel opening [232].…”