Characterization of a 5-HT₃–ELIC Chimera Revealing the Sites of Action of Modulators

Abstract

Cys-loop receptors are major sites of action for many important therapeutically active compounds, but the sites of action of those that do not act at the orthosteric binding site or at the pore are mostly poorly understood. To help understand these, we here describe a chimeric receptor consisting of the extracellular domain of the 5-HTA receptor and the transmembrane domain of a prokaryotic homologue, ELIC. Alterations of some residues at the coupling interface are required for function, but the resulting receptor expresses well and responds to 5-HT with a lower EC (0.34 μM) than that of the 5-HTA receptor. Partial agonists and competitive antagonists of the 5-HTA receptor activate and inhibit the chimera as expected. Examination of a range of receptor modulators, including ethanol, thymol, 5-hydroxyindole, and 5-chloroindole, which can affect the 5-HTA receptor and ELIC, suggest that these compounds act via the transmembrane domain, except for 5-hydroxyindole, which can compete with 5-HT at the orthosteric binding site. The data throw further light on the importance of coupling interface in Cys-loop receptors and provide a platform for examining the mechanism of action of compounds that act in the extracellular domain of the 5-HTA receptor and the transmembrane domain of ELIC.

Large Intracellular Domain-Dependent Effects of Positive Allosteric Modulators on Glycine Receptors

“…The pLGIC receptors show a modular architecture (i.e., ECD, TMD, and ICD). Functional chimeric channels can be formed fusing each of these modules (i.e., ECD + TMDs) from different receptors. ,− We have taken advantage of this approach in order to examine the contribution of each module to the allosteric modulation of the α 1 GlyR. We started with the previously characterized chimera, so-called Lily (G LI C ECD -α 1 G ly R TMD ), which was made by the fusion of the ECD from the prokaryotic homologue GLIC to the TMD of the human α 1 GlyR and contains a small peptide replacing the ICD connecting the TM3 and TM4. , Then, the Lily-ICD chimera was generated by the insertion of the full-length ICD from the α 1 GlyR (Figure A,B).…”

Signal transduction through Cys-loop receptors is mediated by the nonspecific bumping of closely apposed domains

“…It does not seem surprising, then, that defined stretches of amino acids on both the GPCR and the G protein have been found to be critical determinants of this selective interaction (32)(33)(34)(35)(36)(37). It is in this precise context that the reported requirement for the conservation of matching sequences (7,11,13,14,16) or even packing (3,9) or electrostatic properties (15,20,24) across the ECD-TMD interface of pLGICs seemed intriguing to us. Certainly, in contrast to the situation in GPCR signaling (in which case, physiologically aberrant GPCR-G protein pairs need to be avoided) the two modules of pLGICs are part of the same protein, and thus the "correct" combination is inevitable.…”

“…Furthermore, many of the functional ECD-TMD pLGIC chimeras described in the literature contain mutations that make the sequences on both sides of the interface resemble the sequence of one or the other parent (e.g., refs. 9,11,14). Here, instead of aiming to preserve across-the-interface wild-type contacts, we sought to eliminate them.…”

“…to just a few residues, and from packing, "lock-and-key" type of considerations to electrostatic ones (3,9,(13)(14)(15)(16)(17)(18)(19)(20)24).…”

“…The occurrence (in invertebrates) of soluble acetylcholine (ACh)-binding proteins (4) with high homology to the ECD of pLGICs (5) and the finding that fully functional chimeric constructs can often be generated by combining the ECD and TMD of different members of the superfamily (e.g., refs. [6][7][8][9][10][11][12][13][14] make the tightly coupled behavior of these two seemingly independent modules all the more remarkable.…”