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...