Click
here to close Hello! We notice that
you are using Internet Explorer, which is not supported by Echinobase
and may cause the site to display incorrectly. We suggest using a
current version of Chrome,
FireFox,
or Safari.
Proc Natl Acad Sci U S A
2024 Feb 06;1216:e2313853121. doi: 10.1073/pnas.2313853121.
Show Gene links
Show Anatomy links
Loss of activation by GABA in vertebrate delta ionotropic glutamate receptors.
Rosano G
,
Barzasi A
,
Lynagh T
.
???displayArticle.abstract???
Ionotropic glutamate receptors (iGluRs) mediate excitatory signals between cells by binding neurotransmitters and conducting cations across the cell membrane. In the mammalian brain, most of these signals are mediated by two types of iGluRs: AMPA and NMDA (i.e. iGluRs sensitive to 2-amino-3-(5-methyl-3-oxo-1,2-oxazol-4-yl)propanoic acid and N-methyl-D-aspartic acid, respectively). Delta-type iGluRs of mammals also form neurotransmitter-binding channels in the cell membrane, but in contrast, their channel is not activated by neurotransmitter binding, raising biophysical questions about iGluR activation and biological questions about the role of delta iGluRs. We therefore investigated the divergence of delta iGluRs from their iGluR cousins using molecular phylogenetics, electrophysiology, and site-directed mutagenesis. We find that delta iGluRs are found in numerous bilaterian animals (e.g., worms, starfish, and vertebrates) and are closely related to AMPA receptors, both genetically and functionally. Surprisingly, we observe that many iGluRs of the delta family are activated by the classical inhibitory neurotransmitter, γ-aminobutyric acid (GABA). Finally, we identify nine amino acid substitutions that likely gave rise to the inactivity of today's mammalian delta iGluRs, and these mutations abolish activity when engineered into active invertebrate delta iGluRs, partly by inducing receptor desensitization. These results offer biophysical insight into iGluR activity and point to a role for GABA in excitatory signaling in invertebrates.
Fig. 1. GABA-gated channels in the delta iGluR family. (A) Maximum likelihood phylogeny of animal iGluR genes. Selected iGluR branches are colored and labeled. *, genes characterized in panels B–E. X, O, Crassostrea gigas GluD and Saccoglossus kowalevskii GluD, see SI Appendix, Fig. S2. SH-aLRT support for selected branches indicated. Detailed branch support and all gene names in expanded phylogeny, SI Appendix, Fig. S1. (B–E) Example two-electrode voltage clamp recordings of oocytes expressing indicated delta iGluR genes in response to different ligands (Left) and mean ± SEM (n = 4 to 6) normalized concentration-dependent responses (Right). Scale bars: x, 10 s; y, as indicated. (B and C) Wild-type channels were inactive; lurcher-mutant (Lc) DanGlu2A and RatGluD2 and A654C-mutant (AC) RatGluD1 channels were constitutively active. The dashed line indicates zero current; mean responses reflect ligand-induced inhibition of constitutive current normalized to maximum inhibition of constitutive current. (D and E) Wild-type channels were active; mean responses reflect ligand-induced current amplitude normalized to maximum ligand-induced current amplitude. Dan, Danio rerio. Rat, Rattus norvegicus. Aca, Acanthaster planci. Dio, Diopisthoporus longitibus. (F) % amino acid sequence identity of selected delta iGluRs.
Fig. 2. Computational ligand docking. (A and B) Five most energetically favorable binding poses for GABA and D-serine at AcaGluD model (A) and RatGluD2 X-ray structure [PDB:2v3u (13)] (B). (C) Amino acid sequence alignment of selected LBD segments (AcaGluD numbering). (D) Example recording and mean (± SEM, n = 4) normalized responses of indicated mutant AcaGluD receptors to different ligands. Oocytes expressing S713A receptors only responded after concanavalin A treatment (“ConA”, 10 μM).
Fig. 3. Pore properties and pharmacology of AcaGluD receptors. (A) Normalized current (I/I−80 mV) -voltage relationship of GABA-gated currents through AcaGluD-expressing oocytes. Data points mean ± SEM (n = 4), joined by straight lines. (B and C) Example recordings (scale bars x, 5 s; y, 1 μA) and summary data (columns, mean; circles, individual data points) of modulation of GABA- and glutamate-gated current by indicated drugs at AcaGluD-expressing oocytes. (D) Example responses to increasing concentrations of GABA in the absence or presence of extracellular Ca2+ (Left) and normalized (to maximum current in respective condition, “I/Imax”) responses to different agonists (Right, mean ± SEM, n = 5 to 7). (E) Fold enhancement (“ICa2+/IzeroCa2+”) of GABA-gated current by 1.8 mM Ca2+ at oocytes expressing WT or indicated mutant AcaGluD. Inset: example recording from oocyte expressing D558A mutant AcaGluD, scale bars in D and E: x, 10 s; y, 0.15 μA.
Fig. 4. RatGluD2 NTD does not abolish the activity of starfish AcaGluD iGluRs. (A) Cartoon illustrating structural domains of typical iGluR tetramer, one subunit highlighted, showing amino-terminal domain (NTD), ligand-binding domain (LBD), and transmembrane channel domain (TMD). Cell membrane in gray. (B) Amino acid sequence alignment of linker region, illustrating chimera design. Numbering, RatGluD2. (C) Ligand-gated currents in oocytes expressing mutant AcaGluD receptors containing the NTD from RatGluD2 (AcaGluDRatNTD) or the NTD and NTD-LBD linker from RatGluD2 (AcaGluDRatNTDlink). Scale bars: x, 10 s; y, 2 µA. (D) Summarized data from experiments in C.
Fig. 5. RatGluD2-like substitutions impair the ligand-gated activity of AcaGluD receptors. (A) Ligand-gated currents in oocytes expressing indicated mutant AcaGluD receptors or uninjected oocytes. (B) Maximum current amplitude (“Imax”, Upper) and relative ligand-gated current amplitude (“I/Imax”, Lower) and at wild-type (WT) and mutant AcaGluD receptors (mean ± SEM, n = 3 to 5). (C) Immunolabeling c-Myc tag in AcaGluD C-terminal. Upper, example micropgraphs. Lower, Random fluorescence units (RFU) plotted against relative radial distance, peaking at the oocyte surface. White scale bars, 100 μm. (D) AlphaFold model of two adjacent AcaGluD LBDs (rear LBD faded). Selected amino acid residues colored and shown as sticks. Magenta, drastic loss of function; cyan, moderate loss of function. Inset cartoon shows LBDs within full-length receptor and approximate position of F640 residue (SI Appendix, Fig. S6A). (E) Upper, example, and Lower, summarized current responses to 30 mM GABA (“IGABA”) in WT or mutant AcaGluD-expressing oocytes before or after treatment with concanavalin A (“ConA”, 10 μM).
Fig. 6. AcaGluD-like mutations alter RatGluD2 receptor function. (A) D-serine bound RatGluD2 from X-ray structure PDB:2v3u (13). Selected amino acid residues are indicated, colored, shown as sticks, and labeled 5×, 9×, 10×, and/or Lc according to the multiple mutants they were incorporated into. 10× mutant included 10×, 9×, and 5× positions. 9× included 9× and 5×. 5× included only 5×. The Inset cartoon shows LBD within full-length receptor and approximate position of two other residues. (B–E) Example current responses to different neurotransmitter ligands (30 mM) and to pore blocker pentamidine (Pent, 100 μM) in oocytes expressing indicated RatGluD2 mutants. The dashed line indicates zero current baseline. (F) Individual (dots) and mean (columns) responses to ligands in oocytes expressing indicated RatGluD2 mutants (n = 4 to 5).
