Abstract
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.
Highlights
Glycine, the major inhibitory neurotransmitter in the brain stem and spinal cord, contributes to the control of motor pattern generation, the synchronization of spinal reflexes, and the processing of sensory stimuli (Lynch, 2004, 2009; Betz and Laube, 2006; Dutertre et al, 2012)
We found that zGlyRα4a transcripts were present in eye, consistent with a previous report (Hensley et al, 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
Heteromeric zebrafish glycine receptors (zGlyRs) Assemble With a Subunit Stoichiometry of 3α:2ß
Summary
The major inhibitory neurotransmitter in the brain stem and spinal cord, contributes to the control of motor pattern generation, the synchronization of spinal reflexes, and the processing of sensory stimuli (Lynch, 2004, 2009; Betz and Laube, 2006; Dutertre et al, 2012). SS subunits lack the ability to form functional homomeric receptors, due to an absence of critical residues within several N-terminal assembly motifs (Griffon et al, 1999), ß subunits are essential for the synaptic localization of GlyRs owing to the ability of the intracellular loop between transmembranes three and four of ß subunits (ß-loop) to bind to the postsynaptic scaffolding gephyrin (Meyer et al, 1995). This requirement of ß subunits for the synaptic localization of GlyRs has raised questions regarding the subunit stoichiometry of heteromeric GlyRs, with conflicting experimental evidence in support of either 3α:2ß or 2α:3ß (Langosch et al, 1988; Kuhse et al, 1993; Burzomato et al, 2003; Grudzinska et al, 2005; Durisic et al, 2012; Yang et al, 2012)
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