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Front Mol Neurosci
2018 Feb 07;11:286. doi: 10.3389/fnmol.2018.00286.
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Characterization of the Zebrafish Glycine Receptor Family Reveals Insights Into Glycine Receptor Structure Function and Stoichiometry.
Low SE
,
Ito D
,
Hirata H
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To study characterization of zebrafish glycine receptors (zGlyRs), we assessed expression and function of five α- and two ß-subunit encoding GlyR in zebrafish. Our qPCR analysis revealed variable expression during development, while in situ hybridizations uncovered expression in the hindbrain and spinal cord; a finding consistent with the reported expression of GlyR subunits in these tissues from other organisms. Electrophysiological recordings using Xenopus oocytes revealed that all five α subunits form homomeric receptors activated by glycine, and inhibited by strychnine and picrotoxin. In contrast, ß subunits only formed functional heteromeric receptors when co-expressed with α subunits. Curiously, the second transmembranes of both ß subunits were found to lack a phenylalanine at the sixth position that is commonly associated with conferring picrotoxin resistance to heteromeric receptors. Consistent with the absence of phenylalanines at the sixth position, heteromeric zGlyRs often lacked significant picrotoxin resistance. Subsequent efforts revealed that resistance to picrotoxin in both zebrafish and human heteromeric GlyRs involves known residues within transmembrane 2, as well as previously unknown residues within transmembrane 3. We also found that a dominant mutation in human GlyRα1 that gives rise to hyperekplexia, and recessive mutations in zebrafish GlyRßb that underlie the bandoneon family of motor mutants, result in reduced receptor function. Lastly, through the use of a concatenated construct we demonstrate that zebrafish heteromeric receptors assemble with a stoichiometry of 3α:2ß. Collectively, our findings have furthered our knowledge regarding the assembly of heteromeric receptors, and the molecular basis of ß subunit-conferred picrotoxin resistance. These results should aid in future investigations of glycinergic signaling in zebrafish and mammals.
Figure 1. Zebrafish family of glycine receptors (GlyR). (A) Chromosomal (Chr) arrangement of zebrafish GlyR (zGlyR) subunits. Location (in Mbp) are on the right, while name of subunit, length and sequence similarity to human ortholog are given to the left. (B) Phylogenetic analysis of zGlyR subunits. Horizontal scale bar at the bottom reflects amount of genetic change, all other numbers are bootstraps. (mGlyR and hGlyR) mouse and human GlyR subunit. (C) Sequence alignments of zGlyRα subunits to hGlyRα1, and zGlyRß subunits to hGlyRß. Red letters indicate residues conserved among α and ß subunits from zebrafish and human. Blue letters indicate residues conserved between zGlyRß paralogs which differ from hGlyRß. Magenta letters indicate residues conserved between α and ß subunits from zebrafish and human. AD1: assembly domain one, (−)BD and (+)BD: negative and positive faces of the inter-subunit binding sites for glycine and strychnine, M2: transmembrane domain 2. Numbered residues (‘) indicate position with M2.
Figure 3. Whole-mount in situ hybridizations of zGlyR subunits from 48 hours post-fertilization (hpf) larvae. Individual subunits are indicated to the left.
Figure 4. zGlyRα subunits form functional homomeric receptors that are activated by glycine, and inhibited by strychnine and picrotoxin. (A) Two-electrode voltage-clamp recording from an oocyte injected with five femtomoles of zGlyRα1 cRNA exposed to serial application of glycine of increasing amount. Ten oocytes were used for each assay. (B) Cumulative dose-response relationship of glycine-evoked currents. The amplitude of each glycine-evoked response was normalized to the maximally-evoked current for each oocyte (n = 10). Values here and elsewhere represent the average ± SEM. Dashed line denotes EC50. (C,E) Recordings from oocytes exposed to glycine and increasing amounts of strychnine or picrotoxin, respectively (n = 10). (D,F) Cumulative dose-response relationships of strychnine and picrotoxin-blocked currents (n = 10). Extent of blockade was normalized to the current amplitude evoked by 200 μM glycine in each oocyte. Dashed lines denote IC50s.
Figure 5. zGlyRà subunits form functional heteromeric receptors with zGlyRα subunits. Cumulative dose-response relationships of glycine-evoked currents from oocytes co-injected with 2.5 femtomoles of zGlyRα cRNA and 2.5 femtomoles of either zGlyRÃa (A) or zGlyRÃb (B) cRNA. Ten oocytes were used for each assay. (CâF) Cumulative dose-response relationships of strychnine and picrotoxin-blocked currents from heteromeric zGlyRs. Ten oocytes were used for each assay.
Figure 6. Residues within M2 and M3 of zebrafish and human à subunits contribute to picrotoxin resistance. Cumulative effect of amino acid substitutions on picrotoxin-mediated blockade of zGlyRÃa (A), zGlyRÃb (B), and hGlyRà containing receptors (D). TM: triple mutation of residues in M2 of zGlyRà subunits; Ãa: L280F+S286A+S291T, Ãb: L281F+S287A+S292T. Ten oocytes were used for each assay. (C) Sequence alignments of M3 from hGlyRà and zGlyRà subunits. Red and blue letters indicate residues that are conserved between zGlyRà subunits, and zGlyRÃb and hGlyRÃ, respectively.
Figure 7. Mutations in zGlyRßb and zGlyRα1 result in hypomorphic receptors. (A) Location of mutations in zGlyRßb that give rise to bandoneon (beo) mutants, and analogous position in hGlyRα1 of mutations that cause hyperekplexia. (GBM) Gephyrin-binding motif. (B) Cumulative effects of bandoneon mutations on heteromeric receptors’ EC50 for glycine and average 10 mM glycine-evoked currents. Of note, oocytes were injected with 1 femtomole of zGlyRα1 cRNA and four femtomoles of zGlyRßb cRNAs. Ten oocytes were used for each assay. Numbers inside bar here and in (C) represent Hill coefficient, and *p < 0.05, **p < 0.01, and ***p < 0.001. (C) Effect of dominant-negative R271Q mutation on both homomeric and heteromeric receptors’ EC50 for glycine and average 10 mM glycine-evoked currents. Each oocyte was injected with 2.5 femtomoles of venus-tagged zGlyRα1 cRNAs and 2.5 femtomoles of zGlyRßb cRNA. Ten oocytes were used for each assay. (D) Confocal images of HEK293T cells expressing venus-tagged zGlyRα1 subunits.
Figure 8. Heteromeric zGlyRs assemble with a stoichiometry of 3α:2Ã. (A) Schematic detailing the assembly of pentameric zGlyRs from concatemers and either monomeric zGlyRα1 or zGlyRÃb. (B) Two-electrode voltage clamp recordings from oocytes injected with cRNA encoding concatemers alone, or with monomeric zGlyRα1 or zGlyRÃb cRNA. Note that the current scale bars on the top and middle traces differ from the bottom trace. (C) Average glycine EC50 and responses to 10 mM glycine (n = 10). ***p< 0.001.
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Erratum: Characterization of the Zebrafish Glycine Receptor Family Reveals Insights Into Glycine Receptor Structure Function and Stoichiometry.
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