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

Abstract

The Cys-loop family of receptors mediates synaptic neurotransmission in the central nervous system of vertebrates. These receptors share several structural characteristics and assemble in the plasma membrane as multimers with fivefold symmetry. Of these, the ionotropic GABA receptors are key players in the pathogenesis of diseases like epilepsy, anxiety, and schizophrenia. Different experimental approaches have shed some light on the mechanisms behind the function of these receptors; but little is known about their structure at high resolution. Sequence homology with the nicotinic acetylcholine receptor predicts that ionotropic GABA receptors possess four transmembrane segments (TM1-4) and that TM2 forms the wall of the ion channel. However, the role of the other three segments is unclear. The GABAρ1 receptor plays a fundamental role in the regulation of neurotransmission along the visual pathway, is highly sensitive to GABA, and exhibits little desensitization. In our recent investigations of the role of TM4 in receptor function, a key residue in this domain (W475) was found to be involved in activation of the receptor. Here we have generated a structural model of the GABAρ1 receptor in silico and assessed its validity by electrophysiologically testing nine amino acid substitutions of W475 and deletions of the neighboring residues (Y474 and S476). The results identify a critical linkage between the ligand-binding domain and the TM4 domain and provide a framework for more detailed structure-function analyses of ionotropic GABA receptors. GABAρ receptors play a central role in the visual pathway, negatively modulating glutamatergic transmission in the retina (1, 2). These receptors are located at the axon terminals of rod bipolar cells, where GABAergic input is received from amacrine neurons (3). There are three known genes coding for GABAρ receptors, GABAρ1 to -3, all of which form functional homomeric receptors with pharmacological properties very different from those of other GABA receptors. For example, they are highly sensitive to GABA, desensitize very little, and are essentially bicuculline/baclofen insensitive in situ (4, 5) and when expressed in heterologous systems, such as the Xenopus laevis oocyte (6, 7).The Cys-loop receptor superfamily, within which the GABAρ receptors are classified, includes cation-selective receptors, such as the nicotinic acetylcholine (nACh) and 5-hydroxytryptamine 3 (5-HT3) receptors, as well as anion-selective receptors, such as those for glycine and GABA (8). All these receptors share wellconserved amino acid sequences and presumably assemble in the plasma membrane as pentamers. Each subunit of the pentamer contains a large extracellular N-terminal ligand-binding domain (LBD), four transmembrane (TM1-4) helices, and a variable intracellular loop between TM3 and TM4 ( Fig. 1 A and B). The TM2 segment lines the pore domain and has subdomains that selectively discriminate ions according to their electric charge. The remaining three helices (TM1, -3, and -4) function as a "hydr...

Dipicrylamine Modulates GABAρ1 Receptors through Interactions with Residues in the TM4 and Cys-Loop Domains

“…Next, we examined the role of the residue W475 that our model implicated in the charge stabilization of DPA within the hydrophobic pocket of each GABAr1 subunit. As previously demonstrated, the substitution of W475 by electrically charged amino acids (W475R and W475D) produced nonfunctional receptors, indicating the fundamental role of W475 in the gating of the receptor (Estrada-Mondragón et al, 2010). The substitution of W475 with hydrophobic amino acids (W475F, W475L, W475G, and W475A) produced functional channels with less sensitivity and lower efficacy in response to GABA and faster kinetics, and all of the mutants except W475L demonstrated less cooperativity (as reported by the Hill coefficient) than the WT receptor (Table 1).…”

“…Interactions between DPA and In Silico Homomeric LGIC Channels. By using the recently published structure of the anionic b3 homomeric human GABA A R (Miller and Aricescu, 2014), we adjusted the previous GABAr1 model (Estrada-Mondragón et al, 2010), which now exhibits a higher alignment score in homology and identity (Supplemental Figs. 1 and 2).…”

“…Recent progress in the search for the gating mechanism along the channel indicates that the upper half of the pore that is lined by the M2 domain of each subunit undergoes considerable deformation, being functionally coupled through loop M2-M3 with loop 2 of the extracellular domain during ligand-induced activation (Prevost et al, 2012;Unwin and Fujiyoshi, 2012). Lipids contribute in a fundamental way to achieve this coupling through critical points conferred by few hydrophobic residues and by electrically charged residues in such interphases (Fernandez Nievas et al, 2008;daCosta et al, 2009;Estrada-Mondragón et al, 2010). Our previous studies have demonstrated that at least two amino acids that form the hydrophobic cavity for DPA, W475 in TM4 and L207 in the Cys-loop, participate in planar hydrophobic interactions that are necessary for the activation of the GABAr1 channel (Estrada-Mondragón et al, 2010); therefore, the presence of DPA inside the hydrophobic pocket may hinder physiologic TM4-Cys-loop interactions, which in turn alter the gating of the channel (discussed later).…”

New Hyperekplexia Mutations Provide Insight into Glycine Receptor Assembly, Trafficking, and Activation Mechanisms

“…As noted above, we were surprised that the p.E375X subunit was incorporated, given that previous studies have shown that TM4 deletion is incompatible with the surface expression of functional ␣1 GlyRs (32)(33)(34)(35). Indeed, the deletion of only a few residues at the C-terminal end of the TM4 domain is sufficient to render some pLGIC receptors completely nonfunctional (12,44,45).…”

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].…”