eprintid: 10098596 rev_number: 34 eprint_status: archive userid: 608 dir: disk0/10/09/85/96 datestamp: 2020-06-01 10:33:45 lastmod: 2021-11-01 01:51:51 status_changed: 2020-12-18 15:41:11 type: article metadata_visibility: show creators_name: Wu, Z creators_name: Lape, R creators_name: Jopp-Saile, L creators_name: O'Callaghan, BJ creators_name: Greiner, T creators_name: Sivilotti, LG title: The Startle disease mutation, α1S270T, predicts shortening of glycinergic synaptic currents ispublished: pub divisions: UCL divisions: B02 divisions: C08 divisions: D09 divisions: G02 keywords: glycine receptors, human channelopathy, patch-clamp, single channel recording note: © 2020 The Authors. The Journal of Physiology published by John Wiley & Sons Ltd on behalf of The Physiological Society This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. abstract: KEY POINTS: 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 α1 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 GlyR characterised in detail the functional consequences of the α1S270T 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 GlyR (to 0.16; cf. 0.99 for wild-type) but reduced only marginally that of heteromeric GlyR (0.96; cf. 0.99 for wild-type). However, both heteromeric and homomeric mutant GlyR 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 α1S270T 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. This article is protected by copyright. All rights reserved. date: 2020-08-15 official_url: http://dx.doi.org/10.1113/JP279803 oa_status: green full_text_type: other language: eng primo: open primo_central: open_green verified: verified_manual elements_id: 1783692 doi: 10.1113/JP279803 lyricists_name: Lape, Remigijus lyricists_name: O'Callaghan, Benjamin lyricists_name: Sivilotti, Lucia lyricists_id: RLAPE20 lyricists_id: BOCAL97 lyricists_id: LGSIV31 actors_name: Flynn, Bernadette actors_id: BFFLY94 actors_role: owner full_text_status: public publication: The Journal of Physiology volume: 598 number: 16 pagerange: 3417-3438 event_location: England citation: Wu, Z; Lape, R; Jopp-Saile, L; O'Callaghan, BJ; Greiner, T; Sivilotti, LG; (2020) The Startle disease mutation, α1S270T, predicts shortening of glycinergic synaptic currents. The Journal of Physiology , 598 (16) pp. 3417-3438. 10.1113/JP279803 <https://doi.org/10.1113/JP279803>. Green open access document_url: https://discovery.ucl.ac.uk/id/eprint/10098596/7/The%20startle%20disease%20mutation%20%CE%B11S270T%20predicts%20shortening%20of%20glycinergic%20synaptic%20currents.pdf