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The startle disease mutation ?1S270T predicts shortening of glycinergic synaptic currents.

ABSTRACT: KEY POINTS:Loss-of-function mutations in proteins found at glycinergic synapses, most commonly in the ?1 subunit of the glycine receptor (GlyR), cause the startle disease/hyperekplexia channelopathy in man. It was recently proposed that the receptors responsible are presynaptic homomeric GlyRs, rather than postsynaptic heteromeric GlyRs (which mediate glycinergic synaptic transmission), because heteromeric GlyRs are less affected by many startle mutations than homomers. We examined the ?1 startle mutation S270T, at the extracellular end of the M2 transmembrane helix. Recombinant heteromeric GlyRs were less impaired than homomers by this mutation when we measured their response to equilibrium applications of glycine. However, currents elicited by synaptic-like millisecond applications of glycine to outside-out patches were much shorter (7- to 10-fold) in all mutant receptors, both homomeric and heteromeric. Thus, the synaptic function of heteromeric receptors is likely to be impaired by the mutation. ABSTRACT:Human startle disease is caused by mutations in glycine receptor (GlyR) subunits or in other proteins associated with glycinergic synapses. Many startle mutations are known, but it is hard to correlate the degree of impairment at molecular level with the severity of symptoms in patients. It was recently proposed that the disease is caused by disruption in the function of presynaptic homomeric GlyRs (rather than postsynaptic heteromeric GlyRs), because homomeric GlyRs are more sensitive to loss-of-function mutations than heteromers. Our patch-clamp recordings from heterologously expressed GlyRs characterised in detail the functional consequences of the ?1S270T startle mutation, which is located at the extracellular end of the pore lining M2 transmembrane segment (18'). This mutation profoundly decreased the maximum single-channel open probability of homomeric GlyRs (to 0.16; cf. 0.99 for wild type) but reduced only marginally that of heteromeric GlyRs (0.96; cf. 0.99 for wild type). However, both heteromeric and homomeric mutant GlyRs became less sensitive to the neurotransmitter glycine. Responses evoked by brief, quasi-synaptic pulses of glycine onto outside-out patches were impaired in mutant receptors, as deactivation was approximately 10- and 7-fold faster for homomeric and heteromeric GlyRs, respectively. Our data suggest that the ?1S270T mutation is likely to affect the opening step in GlyR activation. The faster decay of synaptic currents mediated by mutant heteromeric GlyRs is expected to reduce charge transfer at the synapse, despite the high equilibrium open probability of these mutant channels.

SUBMITTER: Wu Z

PROVIDER: S-EPMC7649747 | biostudies-literature | 2020 Aug

REPOSITORIES: biostudies-literature

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The startle disease mutation α1S270T predicts shortening of glycinergic synaptic currents.

Wu Zhiyi Z Lape Remigijus R Jopp-Saile Lea L O'Callaghan Benjamin J BJ Greiner Timo T Sivilotti Lucia G LG

The Journal of physiology 20200618 16

<h4>Key points</h4>Loss-of-function mutations in proteins found at glycinergic synapses, most commonly in the α1 subunit of the glycine receptor (GlyR), cause the startle disease/hyperekplexia channelopathy in man. It was recently proposed that the receptors responsible are presynaptic homomeric GlyRs, rather than postsynaptic heteromeric GlyRs (which mediate glycinergic synaptic transmission), because heteromeric GlyRs are less affected by many startle mutations than homomers. We examined the α ...[more]

PMID: 32445491

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Project description:Glycine receptors (GlyRs) are the major mediators of fast synaptic inhibition in the adult human spinal cord and brainstem. Hereditary mutations to GlyRs can lead to the rare, but potentially fatal, neuromotor disorder hyperekplexia. Most mutations located in the large intracellular domain (TM3-4 loop) of the GlyRα1 impair surface expression levels of the receptors. The novel GLRA1 mutation P366L, located in the TM3-4 loop, showed normal surface expression but reduced chloride currents, and accelerated whole-cell desensitization observed in whole-cell recordings. At the single-channel level, we observed reduced unitary conductance accompanied by spontaneous opening events in the absence of extracellular glycine. Using peptide microarrays and tandem MS-based analysis methods, we show that the proline-rich stretch surrounding P366 mediates binding to syndapin I, an F-BAR domain protein involved in membrane remodeling. The disruption of the noncanonical Src homology 3 recognition motif by P366L reduces syndapin I binding. These data suggest that the GlyRα1 subunit interacts with intracellular binding partners and may therefore play a role in receptor trafficking or synaptic anchoring, a function thus far only ascribed to the GlyRβ subunit. Hence, the P366L GlyRα1 variant exhibits a unique set of properties that cumulatively affect GlyR functionality and thus might explain the neuropathological mechanism underlying hyperekplexia in the mutant carriers. P366L is the first dominant GLRA1 mutation identified within the GlyRα1 TM3-4 loop that affects GlyR physiology without altering protein expression at the whole-cell and surface levels.SIGNIFICANCE STATEMENT We show that the intracellular domain of the inhibitory glycine receptor α1 subunit contributes to trafficking and synaptic anchoring. A proline-rich stretch in this receptor domain forms a noncanonical recognition motif important for the interaction with syndapin I (PACSIN1). The disruption of this motif, as present in a human patient with hyperekplexia led to impaired syndapin I binding. Functional analysis revealed that the altered proline-rich stretch determines several functional physiological parameters of the ion channel (e.g., faster whole-cell desensitization) reduced unitary conductance and spontaneous opening events. Thus, the proline-rich stretch from the glycine receptor α1 subunit represents a multifunctional intracellular protein motif.

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The startle disease mutation ?1S270T predicts shortening of glycinergic synaptic currents.

Publications

The startle disease mutation α1S270T predicts shortening of glycinergic synaptic currents.

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