Functional Complementation ofGlra1^spd-ot, a Glycine Receptor Subunit Mutant, by Independently Expressed C-Terminal Domains

Abstract

The oscillator mouse (Glra1 spd-ot ) carries a 9 bp microdeletion plus a 2 bp microinsertion in the glycine receptor ␣1 subunit gene, resulting in the absence of functional ␣1 polypeptides from the CNS and lethality 3 weeks after birth. Depending on differential use of two splice acceptor sites in exon 9 of the Glra1 gene, the mutant allele encodes either a truncated ␣1 subunit (spd ot -trc) or a polypeptide with a C-terminal missense sequence (spd ot -elg). During recombinant expression, both splice variants fail to form ion channels. In complementation studies, a tail construct, encoding the deleted C-terminal sequence, was coexpressed with both mutants. Coexpression with spd ot -trc produced glycine-gated ion channels. Rescue efficiency was increased by inclusion of the wild-type motif RRKRRH. In cultured spinal cord neurons from oscillator homozygotes, viral infection with recombinant C-terminal tail constructs resulted in appearance of endogenous ␣1 antigen. The functional rescue of ␣1 mutants by the C-terminal tail polypeptides argues for a modular subunit architecture of members of the Cys-loop receptor family.

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).…”

Inhibitory Glycine Receptors: An Update

“…Subsequently, additional dominant and recessive inheritance patterns and compound heterozygosity have been described for other patient families in which various GLRA1 missense or null mutations have been identified (4,5,97). Some of these mutations have been found to affect GlyR intracellular trafficking rather than agonist binding or channel gating (69,98). Consistent with an important role of the ␤ subunit in postsynaptic GlyR function, mutations in the GLRB gene have been associated with HKPX2 (97,99).…”

The structural basis of function in Cys-loop receptors

“…These regions also influence receptor expression : the number and characteristics of C-terminal residues in M4 are critical for the expression of 5-HT 3 R on the cell surface (Butler et al 2009) and the expression of non-assembling receptors that contain only the ECD and M1-M3 helices can be rescued by co-expression with M4 (Haeger et al 2010 ;Villmann et al 2009). Thus, the M3 and M4 regions are an integral part of the receptor, and have a function that extends beyond simply shielding M2 from the membrane.…”