De novo GRIN variants in M3 helix associated with neurological disorders control channel gating of NMDA receptor.

N-methyl-D-aspartate receptors (NMDARs) are members of the glutamate receptor family and participate in excitatory postsynaptic transmission throughout the central nervous system. Genetic variants in GRIN genes encoding NMDAR subunits are associated with a spectrum of neurological disorders. The M3 transmembrane helices of the NMDAR couple directly to the agonist-binding domains and form a helical bundle crossing in the closed receptors that occludes the pore. The M3 functions as a transduction element whose conformational change couples ligand binding to opening of an ion conducting pore. In this study, we report the functional consequences of 48 de novo missense variants in GRIN1, GRIN2A, and GRIN2B that alter residues in the M3 transmembrane helix. These de novo variants were identified in children with neurological and neuropsychiatric disorders including epilepsy, developmental delay, intellectual disability, hypotonia and attention deficit hyperactivity disorder. All 48 variants in M3 for which comprehensive testing was completed produce a gain-of-function (28/48) compared to loss-of-function (9/48); 11 variants had an indeterminant phenotype. This supports the idea that a key structural feature of the M3 gate exists to stabilize the closed state so that agonist binding can drive channel opening. Given that most M3 variants enhance channel gating, we assessed the potency of FDA-approved NMDAR channel blockers on these variant receptors. These data provide new insight into the structure-function relationship of the NMDAR gate, and suggest that variants within the M3 transmembrane helix produce a gain-of-function.

Early expression of GluN2A-containing NMDA receptors in a model of fragile X syndrome.

NMDA-type glutamate receptors (NMDARs) play a crucial role in synaptogenesis, circuit development, and synaptic plasticity, serving as fundamental components in cellular models of learning and memory. Their dysregulation has been implicated in several neurological disorders and synaptopathies. NMDARs are heterotetrameric complexes composed of two GluN1 and two GluN2 subunits. The composition of GluN2 subunits determines the main biophysical properties of the channel, such as calcium permeability and gating kinetics, and influences the ability of the receptor to interact with postsynaptic proteins involved in normal synaptic physiology and plasticity, including scaffolding proteins and signaling molecules. During early development, NMDARs in the forebrain contain solely the GluN2B subunit, a necessary subunit for proper synaptogenesis and synaptic plasticity. As the animal matures, the expression of the GluN2A subunit increases, leading to a partial replacement of GluN2B-containing synaptic NMDARs with GluN2A-containing receptors. The switch in the synaptic GluN2A-to-GluN2B ratio has a significant impact on the kinetics of excitatory postsynaptic currents and diminishes the synaptic plasticity capacity. In this study, we present findings indicating that GluN2A expression occurs earlier in a mouse model of fragile X syndrome (FXS). This altered timing of GluN2A expression affects various important parameters of NMDAR-mediated excitatory postsynaptic currents, including maximal current amplitude, decay time, and response to consecutive stimuli delivered in close temporal proximity. These observations suggest that the early expression of GluN2A during a critical period when synapses and circuits are developing could be an underlying factor contributing to the formation of pathological circuits in the FXS mouse model.NEW & NOTEWORTHY NMDA receptors (NMDARs) play important roles in synaptic transmission and are involved in multiple neurological disorders. During development, GluN2A in the forebrain becomes incorporated into previously GluN2B-dominated NMDARs, leading to the "GluN2A/GluN2B ratio switch." This is a crucial step for normal brain development. Here we present findings indicating that GluN2A expression occurs earlier in the fragile X mouse and this could be an underlying factor contributing to the pathology found in the fragile X model.

Clinical features, functional consequences, and rescue pharmacology of missense GRID1 and GRID2 human variants.

GRID1 and GRID2 encode the enigmatic GluD1 and GluD2 proteins, which form tetrameric receptors that play important roles in synapse organization and development of the central nervous system. Variation in these genes has been implicated in neurodevelopmental phenotypes. We evaluated GRID1 and GRID2 human variants from the literature, ClinVar, and clinical laboratories and found that many of these variants reside in intolerant domains, including the amino terminal domain of both GRID1 and GRID2. Other conserved regions, such as the M3 transmembrane domain, show different intolerance between GRID1 and GRID2. We introduced these variants into GluD1 and GluD2 cDNA and performed electrophysiological and biochemical assays to investigate the mechanisms of dysfunction of GRID1/2 variants. One variant in the GRID1 distal amino terminal domain resides at a position predicted to interact with Cbln2/Cbln4, and the variant disrupts complex formation between GluD1 and Cbln2, which could perturb its role in synapse organization. We also discovered that, like the lurcher mutation (GluD2-A654T), other rare variants in the GRID2 M3 domain create constitutively active receptors that share similar pathogenic phenotypes. We also found that the SCHEMA schizophrenia M3 variant GluD1-A650T produced constitutively active receptors. We tested a variety of compounds for their ability to inhibit constitutive currents of GluD receptor variants and found that pentamidine potently inhibited GluD2-T649A constitutive channels (IC50 50 nM). These results identify regions of intolerance to variation in the GRID genes, illustrate the functional consequences of GRID1 and GRID2 variants, and suggest how these receptors function normally and in disease.

Therapeutic potential of N-methyl-D-aspartate receptor modulators in psychiatry.

N-methyl-D-aspartate (NMDA) receptors mediate a slow component of excitatory synaptic transmission, are widely distributed throughout the central nervous system, and regulate synaptic plasticity. NMDA receptor modulators have long been considered as potential treatments for psychiatric disorders including depression and schizophrenia, neurodevelopmental disorders such as Rett Syndrome, and neurodegenerative conditions such as Alzheimer's disease. New interest in NMDA receptors as therapeutic targets has been spurred by the findings that certain inhibitors of NMDA receptors produce surprisingly rapid and robust antidepressant activity by a novel mechanism, the induction of changes in the brain that well outlast the presence of drug in the body. These findings are driving research into an entirely new paradigm for using NMDA receptor antagonists in a host of related conditions. At the same time positive allosteric modulators of NMDA receptors are being pursued for enhancing synaptic function in diseases that feature NMDA receptor hypofunction. While there is great promise, developing the therapeutic potential of NMDA receptor modulators must also navigate the potential significant risks posed by the use of such agents. We review here the emerging pharmacology of agents that target different NMDA receptor subtypes, offering new avenues for capturing the therapeutic potential of targeting this important receptor class.

Epithelial N-methyl-D-aspartate (NMDA) receptors mediate renal vasodilation by affecting kidney autoregulation.

Background: N-methyl-D-aspartate receptor (NMDAR) are amino acid receptors that are well studied in brain physiology; however, their role in kidney is poorly understood. Nonetheless, NMDAR inhibitors can increase serum K+ and reduce GFR, which suggests they have an important physiological role in the kidney. We hypothesized that NMDARs in the distal nephron induce afferent-arteriole vasodilation through the vasodilator mechanism connecting-tubule-glomerular feedback (CNTGF) that involves ENaC activation. Methods and results: Using a tubule-specific transcriptome database combined with molecular biology and microscopy techniques, we showed kidney expression of NMDAR subunits along the nephron and specifically in ENaC-positive cells. This receptor is expressed in both male and female mice, with higher abundance in females (p=0.02). Microperfusing NMDAR agonists into the connecting tubule induced afferent-arteriole vasodilation (EC50 10.7 vs. 24.5 mM; p<0.001) that was blunted or eliminated with the use of NMDAR blocker MK-801 or with the ENaC inhibitor Benzamil, indicating a dependence on CNTGF of the NMDAR-induced vasodilation. In vivo, we confirmed this CNTGF-associated vasodilation using kidney micropuncture (Stop-flow pressure 37.9±2.6 vs. 28.6±1.9 mmHg, NMDAR agonist vs vehicle; p<0.01). We explored NMDAR and ENaC channel interaction by using mpkCCD cells and split-open connecting tubules. We observed increased amiloride-sensitive current following NMDAR activation that was prevented by MK-801 (1.14 vs. 0.4 µAmp; p=0.03). In split-open tubules, NMDAR activation increased ENaC activity (Npo Vehicle vs. NMDA; p=0.04). Conclusion: NMDARs are expressed along the nephron, including ENaC-positive cells, with higher expression in females. Epithelial NMDAR mediates renal vasodilation through the connecting-tubule-glomerular feedback, by increasing ENaC activity.

Clinical and functional consequences of GRIA variants in patients with neurological diseases.

AMPA receptors are members of the glutamate receptor family and mediate a fast component of excitatory synaptic transmission at virtually all central synapses. Thus, their functional characteristics are a critical determinant of brain function. We evaluate intolerance of each GRIA gene to genetic variation using 3DMTR and report here the functional consequences of 52 missense variants in GRIA1-4 identified in patients with various neurological disorders. These variants produce changes in agonist EC50, response time course, desensitization, and/or receptor surface expression. We predict that these functional and localization changes will have important consequences for circuit function, and therefore likely contribute to the patients' clinical phenotype. We evaluated the sensitivity of variant receptors to AMPAR-selective modulators including FDA-approved drugs to explore potential targeted therapeutic options.

Loss of Grin2a causes a transient delay in the electrophysiological maturation of hippocampal parvalbumin interneurons.

N-methyl-D-aspartate receptors (NMDARs) are ligand-gated ionotropic glutamate receptors that mediate a calcium-permeable component to fast excitatory neurotransmission. NMDARs are heterotetrameric assemblies of two obligate GluN1 subunits (GRIN1) and two GluN2 subunits (GRIN2A-GRIN2D). Sequencing data shows that 43% (297/679) of all currently known NMDAR disease-associated genetic variants are within the GRIN2A gene, which encodes the GluN2A subunit. Here, we show that unlike missense GRIN2A variants, individuals affected with disease-associated null GRIN2A variants demonstrate a transient period of seizure susceptibility that begins during infancy and diminishes near adolescence. We show increased circuit excitability and CA1 pyramidal cell output in juvenile mice of both Grin2a+/- and Grin2a-/- mice. These alterations in somatic spiking are not due to global upregulation of most Grin genes (including Grin2b). Deeper evaluation of the developing CA1 circuit led us to uncover age- and Grin2a gene dosing-dependent transient delays in the electrophysiological maturation programs of parvalbumin (PV) interneurons. We report that Grin2a+/+ mice reach PV cell electrophysiological maturation between the neonatal and juvenile neurodevelopmental timepoints, with Grin2a+/- mice not reaching PV cell electrophysiological maturation until preadolescence, and Grin2a-/- mice not reaching PV cell electrophysiological maturation until adulthood. Overall, these data may represent a molecular mechanism describing the transient nature of seizure susceptibility in disease-associated null GRIN2A patients.

Differential responses of disease-related GRIN variants located in pore-forming M2 domain of N-methyl-D-aspartate receptor to FDA-approved inhibitors.

N-methyl-D-aspartate receptors (NMDAR), ionotropic glutamate receptors, mediate a slow component of excitatory synaptic transmission in the central nervous system and play a key role in normal brain function and development. Genetic variations in GRIN genes encoding NMDAR subunits that alter the receptor's functional characteristics are associated with a wide range of neurological and neuropsychiatric conditions. Pathological GRIN variants located in the M2 re-entrant loop lining the channel pore cause significant functional changes, the most consequential alteration being a reduction in voltage-dependent Mg2+ inhibition. Voltage-dependent Mg2+ block is a unique feature of NMDAR biology whereby channel activation requires both ligand binding and postsynaptic membrane depolarization. Thus, loss of NMDAR Mg2+ block will have a profound impact on synaptic function and plasticity. Here, we choose 11 missense variants within the GRIN1, GRIN2A, and GRIN2B genes that alter residues located in the M2 loop and significantly reduce Mg2+ inhibition. Each variant was evaluated for tolerance to genetic variation using the 3-dimensional structure and assessed for functional rescue pharmacology via electrophysiological recordings. Three FDA-approved NMDAR drugs-memantine, dextromethorphan, and ketamine-were chosen based on their ability to bind near the M2 re-entrant loop, potentially rectifying dysregulated NMDAR function by supplementing the reduced voltage-dependent Mg2+ block. These results provide insight of structural determinants of FDA-approved NMDAR drugs at their binding sites in the channel pore and may further define conditions necessary for the use of such agents as potential rescue pharmacology.

De novo CLPTM1 variants with reduced GABAA R current response in patients with epilepsy.

OBJECTIVE: To investigate the clinical features and potential pathogenesis mechanism of de novo CLPTM1 variants associated with epilepsy. METHODS: Identify de novo genetic variants associated with epilepsy by reanalyzing trio-based whole-exome sequencing data. We analyzed the clinical characteristics of patients with these variants and performed functional in vitro studies in cells expressing mutant complementary DNA for these variants using whole-cell voltage-clamp current recordings and outside-out patch-clamp recordings from transiently transfected human embryonic kidney (HEK) cells. RESULTS: Two de novo missense variants related to epilepsy were identified in the CLPTM1 gene. Functional studies indicated that CLPTM1-p.R454H and CLPTM1-p.R568Q variants reduced the γ-aminobutyric acid A receptor (GABAA R) current response amplitude recorded under voltage clamp compared to the wild-type receptors. These variants also reduced the charge transfer and altered the time course of desensitization and deactivation following rapid removal of GABA. The surface expression of the GABAA R γ2 subunit from the CLPTM1-p.R568Q group was significantly reduced compared to CLPTM1-WT. SIGNIFICANCE: This is the first report of functionally relevant variants within the CLPTM1 gene. Patch-clamp recordings showed that these de novo CLPTM1 variants reduce GABAA R currents and charge transfer, which should promote excitation and hypersynchronous activity. This study may provide insights into the molecular mechanisms of the CLPTM1 variants underlying the patients' phenotypes, as well as for exploring potential therapeutic targets for epilepsy.

Development of a Dihydroquinoline-Pyrazoline GluN2C/2D-Selective Negative Allosteric Modulator of the N-Methyl-d-aspartate Receptor.

Subunit-selective inhibition of N-methyl-d-aspartate receptors (NMDARs) is a promising therapeutic strategy for several neurological disorders, including epilepsy, Alzheimer's and Parkinson's disease, depression, and acute brain injury. We previously described the dihydroquinoline-pyrazoline (DQP) analogue 2a (DQP-26) as a potent NMDAR negative allosteric modulator with selectivity for GluN2C/D over GluN2A/B. However, moderate (<100-fold) subunit selectivity, inadequate cell-membrane permeability, and poor brain penetration complicated the use of 2a as an in vivo probe. In an effort to improve selectivity and the pharmacokinetic profile of the series, we performed additional structure-activity relationship studies of the succinate side chain and investigated the use of prodrugs to mask the pendant carboxylic acid. These efforts led to discovery of the analogue (S)-(-)-2i, also referred to as (S)-(-)-DQP-997-74, which exhibits >100- and >300-fold selectivity for GluN2C- and GluN2D-containing NMDARs (IC50 0.069 and 0.035 µM, respectively) compared to GluN2A- and GluN2B-containing receptors (IC50 5.2 and 16 µM, respectively) and has no effects on AMPA, kainate, or GluN1/GluN3 receptors. Compound (S)-(-)-2i is 5-fold more potent than (S)-2a. In addition, compound 2i shows a time-dependent enhancement of inhibitory actions at GluN2C- and GluN2D-containing NMDARs in the presence of the agonist glutamate, which could attenuate hypersynchronous activity driven by high-frequency excitatory synaptic transmission. Consistent with this finding, compound 2i significantly reduced the number of epileptic events in a murine model of tuberous sclerosis complex (TSC)-induced epilepsy that is associated with upregulation of the GluN2C subunit. Thus, 2i represents a robust tool for the GluN2C/D target validation. Esterification of the succinate carboxylate improved brain penetration, suggesting a strategy for therapeutic development of this series for NMDAR-associated neurological conditions.

Classification of missense variants in the N-methyl-d-aspartate receptor GRIN gene family as gain- or loss-of-function.

Advances in sequencing technology have generated a large amount of genetic data from patients with neurological conditions. These data have provided diagnosis of many rare diseases, including a number of pathogenic de novo missense variants in GRIN genes encoding N-methyl-d-aspartate receptors (NMDARs). To understand the ramifications for neurons and brain circuits affected by rare patient variants, functional analysis of the variant receptor is necessary in model systems. For NMDARs, this functional analysis needs to assess multiple properties in order to understand how variants could impact receptor function in neurons. One can then use these data to determine whether the overall actions will increase or decrease NMDAR-mediated charge transfer. Here, we describe an analytical and comprehensive framework by which to categorize GRIN variants as either gain-of-function (GoF) or loss-of-function (LoF) and apply this approach to GRIN2B variants identified in patients and the general population. This framework draws on results from six different assays that assess the impact of the variant on NMDAR sensitivity to agonists and endogenous modulators, trafficking to the plasma membrane, response time course and channel open probability. We propose to integrate data from multiple in vitro assays to arrive at a variant classification, and suggest threshold levels that guide confidence. The data supporting GoF and LoF determination are essential to assessing pathogenicity and patient stratification for clinical trials as personalized pharmacological and genetic agents that can enhance or reduce receptor function are advanced. This approach to functional variant classification can generalize to other disorders associated with missense variants.

GPR37L1 controls maturation and organization of cortical astrocytes during development.

Astrocyte maturation is crucial to proper brain development and function. This maturation process includes the ramification of astrocytic morphology and the establishment of astrocytic domains. While this process has been well-studied, the mechanisms by which astrocyte maturation is initiated are not well understood. GPR37L1 is an astrocyte-specific G protein-coupled receptor (GPCR) that is predominantly expressed in mature astrocytes and has been linked to the modulation of seizure susceptibility in both humans and mice. To investigate the role of GPR37L1 in astrocyte biology, RNA-seq analyses were performed on astrocytes immunopanned from P7 Gpr37L1-/- knockout (L1KO) mouse cortex and compared to those from wild-type (WT) mouse cortex. These RNA-seq studies revealed that pathways involved in central nervous system development were altered and that L1KO cortical astrocytes express lower amounts of mature astrocytic genes compared to WT astrocytes. Immunohistochemical studies of astrocytes from L1KO mouse brain revealed that these astrocytes exhibit overall shorter total process length, and are also less complex and spaced further apart from each other in the mouse cortex. This work sheds light on how GPR37L1 regulates cellular processes involved in the control of astrocyte biology and maturation.

Functional effects of disease-associated variants reveal that the S1-M1 linker of the NMDA receptor critically controls channel opening.

The short pre-M1 helix within the S1-M1 linker (also referred to as the pre-M1 linker) between the agonist-binding domain (ABD, S1) and the M1 transmembrane helix of the NMDA receptor (NMDAR) is devoid of missense variants within the healthy population but is a locus for de novo pathogenic variants associated with neurological disorders. Several de novo variants within this helix have been identified in patients presenting early in life with intellectual disability, developmental delay, and/or epilepsy. In this study, we evaluated functional properties for twenty variants within the pre-M1 linker in GRIN1, GRIN2A, and GRIN2B genes, including six novel missense variants. The effects of pre-M1 variants on agonist potency, sensitivity to endogenous allosteric modulators, response time course, channel open probability, and surface expression were assessed. Our data indicated that virtually all of the variants evaluated altered channel function, and multiple variants had profound functional consequences, which may contribute to the neurological conditions in the patients harboring the variants in this region. These data strongly suggest that the residues within the pre-M1 helix play a key role in channel gating and are highly intolerant to genetic variation.

Novel GluN2B-Selective NMDA Receptor Negative Allosteric Modulator Possesses Intrinsic Analgesic Properties and Enhances Analgesia of Morphine in a Rodent Tail Flick Pain Model.

Many cases of accidental death associated with drug overdose are due to chronic opioid use, tolerance, and addiction. Analgesic tolerance is characterized by a decreased response to the analgesic effects of opioids, requiring increasingly higher doses to maintain the desired level of pain relief. Overactivation of GluN2B-containing N-methyl-d-Aspartate receptors is thought to play a key role in mechanisms underlying cellular adaptation that takes place in the development of analgesic tolerance. Herein, we describe a novel GluN2B-selective negative allosteric modulator, EU93-108, that shows high potency and brain penetrance. We describe the structural basis for binding at atomic resolution. This compound possesses intrinsic analgesic properties in the rodent tail immersion test. EU93-108 has an acute and significant anodyne effect, whereby morphine when combined with EU93-108 produces a higher tail flick latency compared to that of morphine alone. These data suggest that engagement of GluN2B as a target has utility in the treatment of pain, and EU93-108 could serve as an appropriate tool compound to interrogate this hypothesis. Future structure-activity relationship work around this scaffold could give rise to compounds that can be co-administered with opioids to diminish the onset of tolerance due to chronic opioid use, thereby modifying their utility.

Novel neuroactive steroids as positive allosteric modulators of NMDA receptors: mechanism, site of action, and rescue pharmacology on GRIN variants associated with neurological conditions.

N-methyl-D-aspartate receptors (NMDARs) play vital roles in normal brain functions (i.e., learning, memory, and neuronal development) and various neuropathological conditions, such as epilepsy, autism, Parkinson's disease, Alzheimer's disease, and traumatic brain injury. Endogenous neuroactive steroids such as 24(S)-hydroxycholesterol (24(S)-HC) have been shown to influence NMDAR activity, and positive allosteric modulators (PAMs) derived from 24(S)-hydroxycholesterol scaffold can also enhance NMDAR function. This study describes the structural determinants and mechanism of action for 24(S)-hydroxycholesterol and two novel synthetic analogs (SGE-550 and SGE-301) on NMDAR function. We also show that these agents can mitigate the altered function caused by a set of loss-of-function missense variants in NMDAR GluN subunit-encoding GRIN genes associated with neurological and neuropsychiatric disorders. We anticipate that the evaluation of novel neuroactive steroid NMDAR PAMs may catalyze the development of new treatment strategies for GRIN-related neuropsychiatric conditions.

Phase 1 Clinical Results for NP10679, a pH-sensitive GluN2B-selective N-methyl-d-aspartate Receptor Inhibitor.

NP10679 is a context-dependent and subunit-selective negative allosteric modulator of N-methyl-d-aspartate (NMDA) receptors. It is a more potent inhibitor of GluN2B-containing NMDA receptors at the acidic levels of extracellular pH (eg, 6.9) found in the penumbral regions associated with cerebral ischemia than at physiological pH. This property allows NP10679 to act selectively in ischemic tissue while minimizing the nonselective blockade of NMDA receptors in healthy brain, thereby reducing on-target adverse effects. We report the results of a first-in-human pharmacokinetic and safety phase 1 clinical trial in healthy volunteers receiving single or multiple doses of NP10679 (NCT04007263). We found that NP10679 was well-tolerated and with a half-life of 20 hours, which is amenable to once per day dosing. The only notable side effect in this clinical trial was modest somnolence at higher doses, atypical in that the subject could easily be aroused. The overall results suggest that NP10679 is a candidate for further development for use in acute brain injury, such as ischemic stroke or aneurysmal subarachnoid hemorrhage, as well as for use in neuropsychiatric indications.

Overlapping cortical malformations in patients with pathogenic variants in GRIN1 and GRIN2B.

BACKGROUND: Malformations of cortical development (MCDs) have been reported in a subset of patients with pathogenic heterozygous variants in GRIN1 or GRIN2B, genes which encode for subunits of the N-methyl-D-aspartate receptor (NMDAR). The aim of this study was to further define the phenotypic spectrum of NMDAR-related MCDs. METHODS: We report the clinical, radiological and molecular features of 7 new patients and review data on 18 previously reported individuals with NMDAR-related MCDs. Neuropathological findings for two individuals with heterozygous variants in GRIN1 are presented. We report the clinical and neuropathological features of one additional individual with homozygous pathogenic variants in GRIN1. RESULTS: Heterozygous variants in GRIN1 and GRIN2B were associated with overlapping severe clinical and imaging features, including global developmental delay, epilepsy, diffuse dysgyria, dysmorphic basal ganglia and hippocampi. Neuropathological examination in two fetuses with heterozygous GRIN1 variants suggests that proliferation as well as radial and tangential neuronal migration are impaired. In addition, we show that neuronal migration is also impaired by homozygous GRIN1 variants in an individual with microcephaly with simplified gyral pattern. CONCLUSION: These findings expand our understanding of the clinical and imaging features of the 'NMDARopathy' spectrum and contribute to our understanding of the likely underlying pathogenic mechanisms leading to MCD in these patients.

Functional Assessment of Cancer-Linked Mutations in Sensitive Regions of Regulators of G Protein Signaling Predicted by Three-Dimensional Missense Tolerance Ratio Analysis.

Regulators of G protein signaling (RGS) proteins modulate G protein-coupled receptor (GPCR) signaling by acting as negative regulators of G proteins. Genetic variants in RGS proteins are associated with many diseases, including cancers, although the impact of these mutations on protein function is uncertain. Here we analyze the RGS domains of 15 RGS protein family members using a novel bioinformatic tool that measures the missense tolerance ratio (MTR) using a three-dimensional (3D) structure (3DMTR). Subsequent permutation analysis can define the protein regions that are most significantly intolerant (P < 0.05) in each dataset. We further focused on RGS14, RGS10, and RGS4. RGS14 exhibited seven significantly tolerant and seven significantly intolerant residues, RGS10 had six intolerant residues, and RGS4 had eight tolerant and six intolerant residues. Intolerant and tolerant-control residues that overlap with pathogenic cancer mutations reported in the COSMIC cancer database were selected to define the functional phenotype. Using complimentary cellular and biochemical approaches, proteins were tested for effects on GPCR-Gα activation, Gα binding properties, and downstream cAMP levels. Identified intolerant residues with reported cancer-linked mutations RGS14-R173C/H and RGS4-K125Q/E126K, and tolerant RGS14-S127P and RGS10-S64T resulted in a loss-of-function phenotype in GPCR-G protein signaling activity. In downstream cAMP measurement, tolerant RGS14-D137Y and RGS10-S64T and intolerant RGS10-K89M resulted in change of function phenotypes. These findings show that 3DMTR identified intolerant residues that overlap with cancer-linked mutations cause phenotypic changes that negatively impact GPCR-G protein signaling and suggests that 3DMTR is a potentially useful bioinformatics tool for predicting functionally important protein residues. SIGNIFICANCE STATEMENT: Human genetic variant/mutation information has expanded rapidly in recent years, including cancer-linked mutations in regulator of G protein signaling (RGS) proteins. However, experimental testing of the impact of this vast catalogue of mutations on protein function is not feasible. We used the novel bioinformatics tool three-dimensional missense tolerance ratio (3DMTR) to define regions of genetic intolerance in RGS proteins and prioritize which cancer-linked mutants to test. We found that 3DMTR more accurately classifies loss-of-function mutations in RGS proteins than other databases thereby offering a valuable new research tool.

Conserved patterns across ion channels correlate with variant pathogenicity and clinical phenotypes.

Clinically identified genetic variants in ion channels can be benign or cause disease by increasing or decreasing the protein function. As a consequence, therapeutic decision-making is challenging without molecular testing of each variant. Our biophysical knowledge of ion-channel structures and function is just emerging, and it is currently not well understood which amino acid residues cause disease when mutated. We sought to systematically identify biological properties associated with variant pathogenicity across all major voltage and ligand-gated ion-channel families. We collected and curated 3049 pathogenic variants from hundreds of neurodevelopmental and other disorders and 12 546 population variants for 30 ion channel or channel subunits for which a high-quality protein structure was available. Using a wide range of bioinformatics approaches, we computed 163 structural features and tested them for pathogenic variant enrichment. We developed a novel 3D spatial distance scoring approach that enables comparisons of pathogenic and population variant distribution across protein structures. We discovered and independently replicated that several pore residue properties and proximity to the pore axis were most significantly enriched for pathogenic variants compared to population variants. Using our 3D scoring approach, we showed that the strongest pathogenic variant enrichment was observed for pore-lining residues and alpha-helix residues within 5Å distance from the pore axis centre and not involved in gating. Within the subset of residues located at the pore, the hydrophobicity of the pore was the feature most strongly associated with variant pathogenicity. We also found an association between the identified properties and both clinical phenotypes and functional in vitro assays for voltage-gated sodium channels (SCN1A, SCN2A, SCN8A) and N-methyl-D-aspartate receptor (GRIN1, GRIN2A, GRIN2B) encoding genes. In an independent expert-curated dataset of 1422 neurodevelopmental disorder pathogenic patient variants and 679 electrophysiological experiments, we show that pore axis distance is associated with seizure age of onset and cognitive performance as well as differential gain versus loss-of-channel function. In summary, we identified biological properties associated with ion-channel malfunction and show that these are correlated with in vitro functional readouts and clinical phenotypes in patients with neurodevelopmental disorders. Our results suggest that clinical decision support algorithms that predict variant pathogenicity and function are feasible in the future.