Automated Patch Clamp Recordings of GPCR-Gated Ion Channels: Targeting the MC4-R/Kir7.1 Potassium Channel Complex.

Automated patch clamp recording is a valuable technique in drug discovery and the study of ion channels. It allows for the precise measurement and manipulation of channel currents, providing insights into their function and modulation by drugs or other compounds. The melanocortin 4 receptor (MC4-R) is a G protein-coupled receptor (GPCR) crucial to appetite regulation, energy balance, and body weight. MC4-R signaling is complex and involves interactions with other receptors and neuropeptides in the appetite-regulating circuitry. MC4-Rs, like other GPCRs, are known to modulate ion channels such as Kir7.1, an inward rectifier potassium channel, in response to ligand binding. This modulation is critical for controlling ion flow across the cell membrane, which can influence membrane potential, excitability, and neurotransmission. The MC4-R is the target for the anti-obesity drug Imcivree. However, this drug is known to lack optimal potency and also has side effects. Using high-throughput techniques for studying the MC4-R/Kir7.1 complex allows researchers to rapidly screen many compounds or conditions, aiding the development of drugs that target this system. Additionally, automated patch clamp recording of this receptor-channel complex and its ligands can provide valuable functional and pharmacological insights supporting the development of novel therapeutic strategies. This approach can be generalized to other GPCR-gated ion channel functional complexes, potentially accelerating the pace of research in different fields with the promise to uncover previously unknown aspects of receptor-ion channel interactions.

Structure of the Ion Channel Kir7.1 and Implications for its Function in Normal and Pathophysiologic States.

Hereditary defects in the function of the Kir7.1 in the retinal pigment epithelium are associated with the ocular diseases retinitis pigmentosa, Leber congenital amaurosis, and snowflake vitreal degeneration. Studies also suggest that Kir7.1 may be regulated by a GPCR, the melanocortin-4 receptor, in certain hypothalamic neurons. We present the first structures of human Kir7.1 and describe the conformational bias displayed by two pathogenic mutations, R162Q and E276A, to provide an explanation for the basis of disease and illuminate the gating pathway. We also demonstrate the structural basis for the blockade of the channel by a small molecule ML418 and demonstrate that channel blockade in vivo activates MC4R neurons in the paraventricular nucleus of the hypothalamus (PVH), inhibiting food intake and inducing weight loss. Preliminary purification, and structural and pharmacological characterization of an in tandem construct of MC4R and Kir7.1 suggests that the fusion protein forms a homotetrameric channel that retains regulation by liganded MC4R molecules.

Functional coupling between MC4R and Kir7.1 contributes to clozapine-induced hyperphagia.

Most antipsychotic drugs (APDs) induce hyperphagia and weight gain. However, the neural mechanisms are poorly understood, partly due to challenges replicating their metabolic effects in rodents. Here, we report a new mouse model that recapitulates overeating induced by clozapine, a widely prescribed APD. Our study shows that clozapine boosts food intake by inhibiting melanocortin 4 receptor (MC4R) expressing neurons in the paraventricular nucleus of the hypothalamus. Interestingly, neither clozapine nor risperidone, another commonly used APD, affects receptor-ligand binding or the canonical Gαs signaling of MC4R. Instead, they inhibit neuronal activity by enhancing the coupling between MC4R and Kir7.1, leading to the open state of the inwardly rectifying potassium channel. Deletion of Kir7.1 in Mc4r-Cre neurons prevents clozapine-induced weight gain, while treatment with a selective Kir7.1 blocker mitigates overeating in clozapine-fed mice. Our findings unveil a molecular pathway underlying the effect of APDs on feeding behavior and suggest its potential as a therapeutic target.

XOB: A novel phenylalkylamine antagonist of 5-HT2A receptors and voltage-gated sodium channels.

Bipolar disorder impacts millions of patients in the United States but the mechanistic understanding of its pathophysiology and therapeutics is incomplete. Atypical antipsychotic serotonin2A (5-HT2A) receptor antagonists, such as quetiapine and olanzapine, and mood-stabilizing voltage-gated sodium channel (VGSC) blockers, such as lamotrigine, carbamazepine, and valproate, show therapeutic synergy and are often prescribed in combination for the treatment of bipolar disorder. Combination therapy is a complex task for clinicians and patients, often resulting in unexpected difficulties with dosing, drug tolerances, and decreased patient compliance. Thus, an unmet need for bipolar disorder treatment is to develop a therapeutic agent that targets both 5-HT2A receptors and VGSCs. Towards this goal, we developed a novel small molecule that simultaneously antagonizes 5-HT2A receptors and blocks sodium current. The new compound, N-(4-bromo-2,5-dimethoxyphenethyl)-6-(4-phenylbutoxy)hexan-1-amine (XOB) antagonizes 5-HT-stimulated, Gq-mediated, calcium flux at 5-HT2A receptors at low micromolar concentrations while displaying negligible affinity and activity at 5-HT1A, 5-HT2B, and 5-HT2C receptors. At similar concentrations, XOB administration inhibits sodium current in heterologous cells and results in reduced action potential (AP) firing and VGSC-related AP properties in mouse prefrontal cortex layer V pyramidal neurons. Thus, XOB represents a new, proof-of-principle tool that can be used for future preclinical investigations and therapeutic development. This polypharmacology approach of developing a single molecule to act upon two targets, which are currently independently targeted by combination therapies, may lead to safer alternatives for the treatment of psychiatric disorders that are increasingly being found to benefit from the simultaneous targeting of multiple receptors. Significance Statement We synthesized a novel small molecule (XOB) that simultaneously antagonizes two key therapeutic targets of bipolar disorder, 5-HT2A receptors and voltage-gated sodium channels (VGSCs), in heterologous cells, and inhibits the intrinsic excitability of mouse prefrontal cortex layer V pyramidal neurons in brain slices. XOB represents a valuable new proof-of-principle tool for future preclinical investigations and provides a novel molecular approach to the pharmacological treatment of complex neuropsychiatric disease, which often requires a combination of therapeutics for sufficient patient benefit.

Novel Cocrystal Structures of Peptide Antagonists Bound to the Human Melanocortin Receptor 4 Unveil Unexplored Grounds for Structure-Based Drug Design.

Melanocortin 4 receptor (MC4-R) antagonists are actively sought for treating cancer cachexia. We determined the structures of complexes with PG-934 and SBL-MC-31. These peptides differ from SHU9119 by substituting His6 with Pro6 and inserting Gly10 or Arg10. The structures revealed two subpockets at the TM7-TM1-TM2 domains, separated by N2857.36. Two peptide series based on the complexed peptides led to an antagonist activity and selectivity SAR study. Most ligands retained the SHU9119 potency, but several SBL-MC-31-derived peptides significantly enhanced MC4-R selectivity over MC1-R by 60- to 132-fold. We also investigated MC4-R coupling to the K+ channel, Kir7.1. Some peptides activated the channel, whereas others induced channel closure independently of G protein coupling. In cell culture studies, channel activation correlated with increased feeding, while a peptide with Kir7.1 inhibitory activity reduced eating. These results highlight the potential for targeting the MC4-R:Kir7.1 complex for treating positive and restrictive eating disorders.

Epileptic Encephalopathy GABRB Structural Variants Share Common Gating and Trafficking Defects.

Variants in the GABRB gene, which encodes the ß subunit of the GABAA receptor, have been implicated in various epileptic encephalopathies and related neurodevelopmental disorders such as Dravet syndrome and Angelman syndrome. These conditions are often associated with early-onset seizures, developmental regression, and cognitive impairments. The severity and specific features of these encephalopathies can differ based on the nature of the genetic variant and its impact on GABAA receptor function. These variants can lead to dysfunction in GABAA receptor-mediated inhibition, resulting in an imbalance between neuronal excitation and inhibition that contributes to the development of seizures. Here, 13 de novo EE-associated GABRB variants, occurring as missense mutations, were analyzed to determine their impact on protein stability and flexibility, channel function, and receptor biogenesis. Our results showed that all mutations studied significantly impact the protein structure, altering protein stability, flexibility, and function to varying degrees. Variants mapped to the GABA-binding domain, coupling zone, and pore domain significantly impact the protein structure, modifying the ß+/α- interface of the receptor and altering channel activation and receptor trafficking. Our study proposes that the extent of loss or gain of GABAA receptor function can be elucidated by identifying the specific structural domain impacted by mutation and assessing the variability in receptor structural dynamics. This paves the way for future studies to explore and uncover links between the incidence of a variant in the receptor topology and the severity of the related disease.

Targeting the central melanocortin system for the treatment of metabolic disorders.

A large body of preclinical and clinical data shows that the central melanocortin system is a promising therapeutic target for treating various metabolic disorders such as obesity and cachexia, as well as anorexia nervosa. Setmelanotide, which functions by engaging the central melanocortin circuitry, was approved by the FDA in 2020 for use in certain forms of syndromic obesity. Furthermore, the FDA approvals in 2019 of two peptide drugs targeting melanocortin receptors for the treatment of generalized hypoactive sexual desire disorder (bremelanotide) and erythropoietic protoporphyria-associated phototoxicity (afamelanotide) demonstrate the safety of this class of peptides. These approvals have also renewed excitement in the development of therapeutics targeting the melanocortin system. Here, we review the anatomy and function of the melanocortin system, discuss progress and challenges in developing melanocortin receptor-based therapeutics, and outline potential metabolic and behavioural disorders that could be addressed using pharmacological agents targeting these receptors.

GABRG2 Variants Associated with Febrile Seizures.

Febrile seizures (FS) are the most common form of epilepsy in children between six months and five years of age. FS is a self-limited type of fever-related seizure. However, complicated prolonged FS can lead to complex partial epilepsy. We found that among the GABAA receptor subunit (GABR) genes, most variants associated with FS are harbored in the γ2 subunit (GABRG2). Here, we characterized the effects of eight variants in the GABAA receptor γ2 subunit on receptor biogenesis and channel function. Two-thirds of the GABRG2 variants followed the expected autosomal dominant inheritance in FS and occurred as missense and nonsense variants. The remaining one-third appeared as de novo in the affected probands and occurred only as missense variants. The loss of GABAA receptor function and dominant negative effect on GABAA receptor biogenesis likely caused the FS phenotype. In general, variants in the GABRG2 result in a broad spectrum of phenotypic severity, ranging from asymptomatic, FS, genetic epilepsy with febrile seizures plus (GEFS+), and Dravet syndrome individuals. The data presented here support the link between FS, epilepsy, and GABRG2 variants, shedding light on the relationship between the variant topological occurrence and disease severity.

The unique structural characteristics of the Kir 7.1 inward rectifier potassium channel: a novel player in energy homeostasis control.

The inward rectifier potassium channel Kir7.1, encoded by the KCNJ13 gene, is a tetramer composed of two-transmembrane domain-spanning monomers, closer in homology to Kir channels associated with potassium transport such as Kir1.1, 1.2, and 1.3. Compared with other channels, Kir7.1 exhibits small unitary conductance and low dependence on external potassium. Kir7.1 channels also show a phosphatidylinositol 4,5-bisphosphate (PIP2) dependence for opening. Accordingly, retinopathy-associated Kir7.1 mutations mapped at the binding site for PIP2 resulted in channel gating defects leading to channelopathies such as snowflake vitreoretinal degeneration and Leber congenital amaurosis in blind patients. Lately, this channel's role in energy homeostasis was reported due to the direct interaction with the melanocortin type 4 receptor (MC4R) in the hypothalamus. As this channel seems to play a multipronged role in potassium homeostasis and neuronal excitability, we will discuss what is predicted from a structural viewpoint and its possible implications for hunger control.

Development of an automated screen for Kv7.2 potassium channels and discovery of a new agonist chemotype.

To identify pore domain ligands on Kv7.2 potassium ion channels, we compared wild-type (WT) and W236L mutant Kv7.2 channels in a series of assays with previously validated and novel agonist chemotypes. Positive controls were retigabine, flupirtine, and RL-81; i.e. Kv7.2 channel activators that significantly shift voltage-dependent activation to more negative potentials (ΔV50) at 5 µM. We identified 6 new compounds that exhibited differential enhancing activity between WT and W236L mutant channels. Whole cell patch-clamp electrophysiology studies were conducted to identify Kv7.2. Kv7.2/3, Kv7.4, and Kv7.5 selectivity. Our results validate the SyncroPatch platform and establish new structure activity relationships (SAR). Specifically, in addition to selective Kv7.2, Kv7.2/3, Kv7.4. and Kv7.5 agonists, we identified a novel chemotype, ZK-21, a 4-aminotetrahydroquinoline that is distinct from any of the previously described Kv7 channel modifiers. Using flexible receptor docking, ZK-21 was predicted to be stabilized by W236 and bind perpendicular to retigabine, burying the benzyl carbamate group into a tunnel reaching the core of the pore domain.

Dravet syndrome-associated mutations in GABRA1, GABRB2 and GABRG2 define the genetic landscape of defects of GABAA receptors.

Dravet syndrome is a rare, catastrophic epileptic encephalopathy that begins in the first year of life, usually with febrile or afebrile hemiclonic or generalized tonic-clonic seizures followed by status epilepticus. De novo variants in genes that mediate synaptic transmission such as SCN1A and PCDH19 are often associated with Dravet syndrome. Recently, GABAA receptor subunit genes (GABRs) encoding α1 (GABRA1), ß3 (GABRB3) and γ2 (GABRG2), but not ß2 (GABRB2) or ß1 (GABRB1), subunits are frequently associated with Dravet syndrome or Dravet syndrome-like phenotype. We performed next generation sequencing on 870 patients with Dravet syndrome and identified nine variants in three different GABRs. Interestingly, the variants were all in genes encoding the most common GABAA receptor, the α1ß2γ2 receptor. Mutations in GABRA1 (c.644T>C, p. L215P; c.640C>T, p. R214C; c.859G>A; V287I; c.641G>A, p. R214H) and GABRG2 (c.269C>G, p. T90R; c.1025C>T, p. P342L) presented as de novo cases, while in GABRB2 two variants were de novo (c.992T>C, p. F331S; c.542A>T, p. Y181F) and one was autosomal dominant and inherited from the maternal side (c.990_992del, p.330_331del). We characterized the effects of these GABR variants on GABAA receptor biogenesis and channel function. We found that defects in receptor gating were the common deficiency of GABRA1 and GABRB2 Dravet syndrome variants, while mainly trafficking defects were found with the GABRG2 (c.269C>G, p. T90R) variant. It seems that variants in α1 and ß2 subunits are less tolerated than in γ2 subunits, since variant α1 and ß2 subunits express well but were functionally deficient. This suggests that all of these GABR variants are all targeting GABR genes that encode the assembled α1ß2γ2 receptor, and regardless of which of the three subunits are mutated, variants in genes coding for α1, ß2 and γ2 receptor subunits make them candidate causative genes in the pathogenesis of Dravet syndrome.

Membrane orientation and oligomerization of the melanocortin receptor accessory protein 2.

The melanocortin receptor accessory protein 2 (MRAP2) plays a pivotal role in the regulation of several G protein-coupled receptors that are essential for energy balance and food intake. MRAP2 loss-of-function results in obesity in mammals. MRAP2 and its homolog MRAP1 have an unusual membrane topology and are the only known eukaryotic proteins that thread into the membrane in both orientations. In this study, we demonstrate that the conserved polybasic motif that dictates the membrane topology and dimerization of MRAP1 does not control the membrane orientation and dimerization of MRAP2. We also show that MRAP2 dimerizes through its transmembrane domain and can form higher-order oligomers that arrange MRAP2 monomers in a parallel orientation. Investigating the molecular details of MRAP2 structure is essential for understanding the mechanism by which it regulates G protein-coupled receptors and will aid in elucidating the pathways involved in metabolic dysfunction.

Determination of the melanocortin-4 receptor structure identifies Ca2+ as a cofactor for ligand binding.

The melanocortin-4 receptor (MC4R) is involved in energy homeostasis and is an important drug target for syndromic obesity. We report the structure of the antagonist SHU9119-bound human MC4R at 2.8-angstrom resolution. Ca2+ is identified as a cofactor that is complexed with residues from both the receptor and peptide ligand. Extracellular Ca2+ increases the affinity and potency of the endogenous agonist α-melanocyte-stimulating hormone at the MC4R by 37- and 600-fold, respectively. The ability of the MC4R crystallized construct to couple to ion channel Kir7.1, while lacking cyclic adenosine monophosphate stimulation, highlights a heterotrimeric GTP-binding protein (G protein)-independent mechanism for this signaling modality. MC4R is revealed as a structurally divergent G protein-coupled receptor (GPCR), with more similarity to lipidic GPCRs than to the homologous peptidic GPCRs.

The GPCR accessory protein MRAP2 regulates both biased signaling and constitutive activity of the ghrelin receptor GHSR1a.

Ghrelin is a hormone secreted by the stomach during fasting periods and acts through its receptor, the growth hormone secretagogue 1a (GHSR1a), to promote food intake and prevent hypoglycemia. As such, GHSR1a is an important regulator of energy and glucose homeostasis and a target for the treatment of obesity. Here, we showed that the accessory protein MRAP2 altered GHSR1a signaling by inhibiting its constitutive activity, as well as by enhancing its G protein-dependent signaling and blocking the recruitment and signaling of ß-arrestin in response to ghrelin. In addition, the effects of MRAP2 on the Gαq and ß-arrestin pathways were independent and involved distinct regions of MRAP2. These findings may have implications for the regulation of ghrelin function in vivo and the role of MRAP2 in energy homeostasis. They also show that accessory proteins can bias signaling downstream of GPCRs in response to their endogenous agonist.

Altered inhibitory synapses in de novo GABRA5 and GABRA1 mutations associated with early onset epileptic encephalopathies.

We performed next generation sequencing on 1696 patients with epilepsy and intellectual disability using a gene panel with 480 epilepsy-related genes including all GABAA receptor subunit genes (GABRs), and we identified six de novo GABR mutations, two novel GABRA5 mutations (c.880G>T, p.V294F and c.1238C>T, p.S413F), two novel GABRA1 mutations (c.778C>T, p.P260S and c.887T>C, p.L296S/c.944G>T, p.W315L) and two known GABRA1 mutations (c.335G>A, p.R112Q and c.343A>G, p.N115D) in six patients with intractable early onset epileptic encephalopathy. The α5(V294F and S413F) and α1(P260S and L296S/W315L) subunit residue substitutions were all in transmembrane domains, while the α1(R112Q and N115R) subunit residue substitutions were in the N-terminal GABA binding domain. Using multidisciplinary approaches, we compared effects of mutant GABAA receptor α5 and α1 subunits on the properties of recombinant α5ß3γ2 and α1ß3γ2 GABAA receptors in both neuronal and non-neuronal cells and characterized their effects on receptor clustering, biogenesis and channel function. GABAA receptors containing mutant α5 and α1 subunits all had reduced cell surface and total cell expression with altered endoplasmic reticulum processing, impaired synaptic clustering, reduced GABAA receptor function and decreased GABA binding potency. Our study identified GABRA5 as a causative gene for early onset epileptic encephalopathy and expands the mutant GABRA1 phenotypic spectrum, supporting growing evidence that defects in GABAergic neurotransmission contribute to early onset epileptic encephalopathy phenotypes.

A structural look at GABAA receptor mutations linked to epilepsy syndromes.

Understanding the genetic variation in GABAA receptor subunit genes (GABRs), GABRA1-6, GABRB1-3, GABRG1-3 and GABRD, in individuals affected by epilepsy may improve the diagnosis and treatment of epilepsy syndromes through identification of disease-associated variants. However, the lack of functional analysis and validation of many novel and previously reported familial and de novo mutations have made it challenging to address meaningful gene associations with epilepsy syndromes. GABAA receptors belong to the Cys-loop receptor family. Even though GABAA receptor mutant residues are widespread among different GABRs, their frequent occurrence in important structural domains that share common functional features suggests associations between structure and function.

Phosphatidylinositol 4,5-bisphosphate (PIP2) regulates KCNQ3 K+ channels by interacting with four cytoplasmic channel domains.

Phosphatidylinositol 4,5-bisphosphate (PIP2) in the plasma membrane regulates the function of many ion channels, including M-type (potassium voltage-gated channel subfamily Q member (KCNQ), Kv7) K+ channels; however, the molecular mechanisms involved remain unclear. To this end, we here focused on the KCNQ3 subtype that has the highest apparent affinity for PIP2 and performed extensive mutagenesis in regions suggested to be involved in PIP2 interactions among the KCNQ family. Using perforated patch-clamp recordings of heterologously transfected tissue culture cells, total internal reflection fluorescence microscopy, and the zebrafish (Danio rerio) voltage-sensitive phosphatase to deplete PIP2 as a probe, we found that PIP2 regulates KCNQ3 channels through four different domains: 1) the A-B helix linker that we previously identified as important for both KCNQ2 and KCNQ3, 2) the junction between S6 and the A helix, 3) the S2-S3 linker, and 4) the S4-S5 linker. We also found that the apparent strength of PIP2 interactions within any of these domains was not coupled to the voltage dependence of channel activation. Extensive homology modeling and docking simulations with the WT or mutant KCNQ3 channels and PIP2 were consistent with the experimental data. Our results indicate that PIP2 modulates KCNQ3 channel function by interacting synergistically with a minimum of four cytoplasmic domains.

G protein-coupled receptors differentially regulate glycosylation and activity of the inwardly rectifying potassium channel Kir7.1.

Kir7.1 is an inwardly rectifying potassium channel with important roles in the regulation of the membrane potential in retinal pigment epithelium, uterine smooth muscle, and hypothalamic neurons. Regulation of G protein-coupled inwardly rectifying potassium (GIRK) channels by G protein-coupled receptors (GPCRs) via the G protein ßγ subunits has been well characterized. However, how Kir channels are regulated is incompletely understood. We report here that Kir7.1 is also regulated by GPCRs, but through a different mechanism. Using Western blotting analysis, we observed that multiple GPCRs tested caused a striking reduction in the complex glycosylation of Kir7.1. Further, GPCR-mediated reduction of Kir7.1 glycosylation in HEK293T cells did not alter its expression at the cell surface but decreased channel activity. Of note, mutagenesis of the sole Kir7.1 glycosylation site reduced conductance and open probability, as indicated by single-channel recording. Additionally, we report that the L241P mutation of Kir7.1 associated with Lebers congenital amaurosis (LCA), an inherited retinal degenerative disease, has significantly reduced complex glycosylation. Collectively, these results suggest that Kir7.1 channel glycosylation is essential for function, and this activity within cells is suppressed by most GPCRs. The melanocortin-4 receptor (MC4R), a GPCR previously reported to induce ligand-regulated activity of this channel, is the only GPCR tested that does not have this effect on Kir7.1.

A Missense Mutation A384P Associated with Human Hyperekplexia Reveals a Desensitization Site of Glycine Receptors.

Hyperekplexia, an inherited neuronal disorder characterized by exaggerated startle responses with unexpected sensory stimuli, is caused by dysfunction of glycinergic inhibitory transmission. From analysis of newly identified human hyperekplexia mutations in the glycine receptor (GlyR) α1 subunit, we found that an alanine-to-proline missense mutation (A384P) resulted in substantially higher desensitization level and lower agonist sensitivity of homomeric α1 GlyRs when expressed in HEK cells. The incorporation of the ß subunit fully reversed the reduction in agonist sensitivity and partially reversed the desensitization of α1A384P The heteromeric α1A384Pß GlyRs showed enhanced desensitization but unchanged agonist-induced maximum responses, surface expression, main channel conductance, and voltage dependence compared with that of the wild-type α1ß (α1WTß) GlyRs. Coexpression of the R392H and A384P mutant α1 subunits, which mimic the expression of the compound heterozygous mutation in a hyperekplexia patient, resulted in channel properties similar to those with α1A384P subunit expression alone. In comparison, another human hyperekplexia mutation α1P250T, which was previously reported to enhance desensitization, caused a strong reduction in maximum currents in addition to the altered desensitization. These results were further confirmed by overexpression of α1P250T or α1A384P subunits in cultured neurons isolated from SD rats of either sex. Moreover, the IPSC-like responses of cells expressing α1A384Pß induced by repeated glycine pulses showed a stronger frequency-dependent reduction than those expressing α1WTß. Together, our findings demonstrate that A384 is associated with the desensitization site of the α1 subunit and its proline mutation produced enhanced desensitization of GlyRs, which contributes to the pathogenesis of human hyperekplexia.SIGNIFICANCE STATEMENT Human startle disease is caused by impaired synaptic inhibition in the brainstem and spinal cord, which is due to either direct loss of GlyR channel function or reduced number of synaptic GlyRs. Considering that fast decay kinetics of GlyR-mediated inhibitory synaptic responses, the question was raised whether altered desensitization of GlyRs will cause dysfunction of glycine transmission and disease phenotypes. Here, we found that the α1 subunit mutation A384P, identified from startle disease patients, results in enhanced desensitization and leads to rapidly decreasing responses in the mutant GlyRs when they are activated repeatedly by the synaptic-like simulation. These observations suggest that the enhanced desensitization of postsynaptic GlyRs could be the primary pathogenic mechanism of human startle disease.