The frameshift mutation oscillator (Glra1spd-ot) produces a complete loss of glycine receptor α1-polypeptide in mouse central nervous system

New Hyperekplexia Mutations Provide Insight into Glycine Receptor Assembly, Trafficking, and Activation Mechanisms

“…The reduced glycine sensitivity was only partly compensated by co-expressing the ␤ wild type subunit. This result strongly suggests that the truncated subunit is incorporated into functional receptors, which is unexpected given that previous GlyR studies indicated that TM4 deletion is incompatible with the surface expression of functional ␣1 GlyRs (32)(33)(34)(35). Given our unexpected result, we sought to confirm whether the p.E375X mutant subunit was incorporated into functional GlyRs using voltage clamp fluorometry.…”

“…We also attempted to confirm it using immunofluorescence, but the incorporation rate may have been too low to allow surface expression to be detected. As noted above, we were surprised that the p.E375X subunit was incorporated, given that previous studies have shown that TM4 deletion is incompatible with the surface expression of functional ␣1 GlyRs (32)(33)(34)(35). Indeed, the deletion of only a few residues at the C-terminal end of the TM4 domain is sufficient to render some pLGIC receptors completely nonfunctional (12,44,45).…”

The Importance of TM3-4 Loop Subdomains for Functional Reconstitution of Glycine Receptors by Independent Domains

“…For GlyR, nonsense and frameshift mutations have been described in patients suffering from hyperekplexia, a rare neuromotor disorder (20,26). A similar hyperekplexia-like phenotype was observed in the GlyR mouse mutant oscillator, resulting from lack of GlyR expression due to introduction of a premature stop codon at amino acid position 355 (21). Loss of receptor proteins has also been found in other channelopathies, such as GEFSϩ (generalized epilepsy with febrile seizures plus) associated with truncated GABA A receptor variants (27).…”

“…TM4 of the GlyR-Truncated variants of the GlyR ␣1 subunit, which have been identified in a family with hyperekplexia (20) and are present in the mouse model oscillator (21), lacking most of the intracellular TM3-4 loop, the TM4, and the C terminus (␣1-trc representing residues 1-357), are not able to gate Cl Ϫ currents. Coexpressions with a corresponding complementation construct iD-TM4, led to reconstitution of functional ion channels (14).…”

Conserved High Affinity Ligand Binding and Membrane Association in the Native and Refolded Extracellular Domain of the Human Glycine Receptor α1-Subunit

“…Cover slips were transferred to slides and examined on a confocal microscope (Leica, Bensheim, Germany). (23). For all methods, dried filters were subjected to scintillation counting (Roth, Karlsruhe, Germany).…”