Investigation Of GlyR And GABA_AR Interaction In Startle Disease

Hyperekplexia is a rare nonepileptic neuologic disorder,characterized by exaggerated startle responses and muscle stiffness following an unexpected stimulus,genetically caused by mutation of Glycine receptor(GlyR).Multiple hyperekplexia related mutations of GlyRal subunit,such as R271Q,S267Q and Q266I has been reported in several clinical investigations and animal studies.GlyR,as one of the major inhibitory neurotransmitter receptors,is widely distributed in the central nervous system,especially in brainstem and spinal cord.GlyR may play important effect in keeping the balance of excitory and inhibitory system.GABAAR,belonging to the Cys-loop ligand-gated ion channel(LGIC)superfamiliy as well as GlyR,is another major inhibitory neurotransmitter receptor.Similarly,GABAAR also located on pre-,post-and extra-synaptic membrane of a synapse,depending on the composition of different GABAAR subunits.Inhyperekplexia disease,whether potential compensatory mechanism through GABAAR subunits happened is remained unclear.In this study,we checked function of GABAAR in several hyperekplexia mice.We collect and measured GABA release and GABAAR functioning using brain slice patchclam recording in GlyRα1R271Q and GlyRα1S267Q hyperekplexia mice.GABAergic spontaneous inhibitory postsynaptic frequency and amplitude(sIPSC)was remarkably attenuated,which indicated that dysfunction,instead of compensatory effect GABAAR in hyperekpelxia mice.Next,we investigated whether the GlyRal mutations could cause GABAAR dysfunction when co-expressed in HEK-293T cells.Only specific GlyRα1 mutations could induced GABAAR deficiency.One possible mechanism for GABAAR deficiency in presence of mutant GlyRal is that there may exist a direct interaction between these two proteins.So we conducted co-immunprecipitation(Co-IP)assay and we found R271Q and S267Q GlyRal mutants had a stronger interaction with GABAAR when co-expressed in HEK-293T cell.Similar results were also observed in hyperekpelxia mice carrying GlyRal mutations.To further explore the molecular insight into the role of site R271 in association between GABAAR and GlyR,we constructed 9 other mutations of R271 site.We foundonly specific GlyRα1 mutations caused dysfunction of GABAAR.Interestingly,we found that GlyR β subunit rescued both function of GlyRα1 mutants and GABAAR through interrupted the association between GlyRal mutants and GABAARs.Meanwhile,in brainstem slice,we found function of GABAAR(α5β2γ2),which almost located at pre-and extra-synaptic,was remarkably reduced.Strong binding between GlyR mutants and GABAAR was also observed in GlyRα1S267Q mice.Benzodezepine has been used as the first linemedicineto treat hyperekpledixia patients in clinics.We found diazepam rescued both mIPSCs frequency and bicuculline-sensitive tonic current and these effect of diazepam were remarkably blocked by Xli 093,an inhibitor specifically blocking diazepam induced potentiation on a5-containing GABAAR.Next,we found diazepam could also rescue startle response of GlyRα1S267Q mice and these effect could be diminished by intra-brainstem microinjection Xli 093.Emerging evidence suggests that GABAARα5 subunits are mainly localized in pre-and extra-synaptic sites in several brain regions,including the hippocampus,spinal cord and brainstem.Therefore dysfunction of GABAAR maybe another mechanism causing hyperekplexia disease,and GABAARa5 specific agonist or modulator maybe a potential therapeutic target for hyperepleixa disease.We also combined modifiedpatchclamp technique and modified mass spectrometry to detect molecular from single neuron cytoplasm in brain slice of mice,directly.This situ sampling skill could not onlymeasure the biological activities of the single neuron but also detect cytoplasmic constituents.We detect four brain regions using this new skill and confrmed traditional Gln-GABA pathway.