A novel hyperekplexia-causing mutation in the pre-transmembrane segment 1 of the human glycine receptor alpha1 subunit reduces membrane expression and impairs gating by agonists
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Abstract
In this study, we have compared the functional consequences
of three mutations (R218Q, V260M, and Q266H)
in the 1 subunit of the glycine receptor (GlyRA1) causing
hyperekplexia, an inherited neurological channelopathy.
In HEK-293 cells, the agonist EC50s for glycine-
activated Cl currents were increased from 26 M
in wtGlyRA1, to 5747, 135, and 129 M in R218Q, V260M,
and Q266H GlyRA1 channels, respectively. Cl currents
elicited by -alanine and taurine, which behave as agonists
at wtGlyRA1, were decreased in V260M and Q266H
mutant receptors and virtually abolished in GlyRA1
R218Q receptors. Gly-gated Cl currents were similarly
antagonized by low concentrations of strychnine in both
wild-type (wt) and R218Q GlyRA1 channels, suggesting
that the Arg-218 residue plays a crucial role in GlyRA1
channel gating, with only minor effects on the agonist/
antagonist binding site, a hypothesis supported by our
molecular model of the GlyRA1 subunit. The R218Q
mutation, but not the V260M or the Q266H mutation,
caused a marked decrease of receptor subunit expression
both in total cell lysates and in isolated plasma
membrane proteins. This decreased expression does
not seem to explain the reduced agonist sensitivity of
GlyRA1 R218Q channels since no difference in the apparent
sensitivity to glycine or taurine was observed
when wtGlyRA1 receptors were expressed at levels
comparable with those of R218Q mutant receptors. In
conclusion, multiple mechanisms may explain the dramatic
decrease in GlyR function caused by the R218Q
mutation, possibly providing the molecular basis for
its association with a more severe clinical phenotype