Evolutionary conservation of putative suicidality-related risk genes that produce diminished motivation corrected by clozapine, lithium and antidepressants.

Background: Genome wide association studies (GWAS) and candidate gene analyses have identified genetic variants and genes that may increase the risk for suicidal thoughts and behaviors (STBs). Important unresolved issues surround these tentative risk variants such as the characteristics of the associated genes and how they might elicit STBs. Methods: Putative suicidality-related risk genes (PSRGs) were identified by comprehensive literature search and were characterized with respect to evolutionary conservation, participation in gene interaction networks and associated phenotypes. Evolutionary conservation was established with database searches and BLASTP queries, whereas gene-gene interactions were ascertained with GeneMANIA. We then examined whether mutations in risk-gene counterparts in C. elegans produced a diminished motivation phenotype previously connected to suicide risk factors. Results and conclusions: From the analysis, 105 risk-gene candidates were identified and found to be: 1) highly conserved during evolution, 2) enriched for essential genes, 3) involved in significant gene-gene interactions, and 4) associated with psychiatric disorders, metabolic disturbances and asthma/allergy. Evaluation of 17 mutant strains with loss-of-function/deletion mutations in PSRG orthologs revealed that 11 mutants showed significant evidence of diminished motivation that manifested as immobility in a foraging assay. Immobility was corrected in some or all of the mutants with clozapine, lithium and tricyclic antidepressant drugs. In addition, 5-HT2 receptor and muscarinic receptor antagonists restored goal-directed behavior in most or all of the mutants. These studies increase confidence in the validity of the PSRGs and provide initial clues about possible mechanisms that mediate STBs.

Role of Plasma Membrane Dicarboxylate Transporters in the Uptake and Toxicity of Diglycolic Acid, a Metabolite of Diethylene Glycol, in Human Proximal Tubule Cells.

Diethylene glycol (DEG) mass poisonings have resulted from ingestion of pharmaceuticals mistakenly adulterated with DEG, typically leading to proximal tubular necrosis and acute kidney injury. The metabolite, diglycolic acid (DGA) accumulates greatly in kidney tissue and its direct administration results in toxicity identical to that in DEG-treated rats. DGA is a dicarboxylic acid, similar in structure to metabolites like succinate. These studies have assessed the mechanism for cellular accumulation of DGA, specifically whether DGA is taken into primary cultures of human proximal tubule (HPT) cells via sodium dicarboxylate transporters (NaDC-1 or NaDC-3) like those responsible for succinate uptake. When HPT cells were cultured on membrane inserts, sodium-dependent succinate uptake was observed from both apical and basolateral directions. Pretreatment with the NaDC-1 inhibitor N-(p-amylcinnamoyl)anthranilic acid (ACA) markedly reduced apical uptakes of both succinate and DGA. Basolateral uptake of both succinate and DGA were decreased similarly following combined treatment with ACA and the NaDC-3 inhibitor 2,3-dimethylsuccinate. When the cells were pretreated with siRNA to knockdown NaDC-1 function, apical uptake of succinate and toxicity of apically applied DGA were reduced, while the reduction in basolateral succinate uptake and basolateral DGA toxicity was marginal with NaDC-3 knockdown. DGA reduced apical uptake of succinate but not basolateral uptake. This study confirmed that primary HPT cells retain sodium dicarboxylate transport functionality and that DGA was taken up by these transporters. This study identified NaDC-1 as a likely and NaDC-3 as a possible molecular target to reduce uptake of this toxic metabolite by the kidney.

How Variation in Risk Allele Output and Gene Interactions Shape the Genetic Architecture of Schizophrenia.

Schizophrenia is a highly heritable polygenic psychiatric disorder. Characterization of its genetic architecture may lead to a better understanding of the overall burden of risk variants and how they determine susceptibility to disease. A major goal of this project is to develop a modeling approach to compare and quantify the relative effects of single nucleotide polymorphisms (SNPs), copy number variants (CNVs) and other factors. We derived a mathematical model for the various genetic contributions based on the probability of expressing a combination of risk variants at a frequency that matched disease prevalence. The model included estimated risk variant allele outputs (VAOs) adjusted for population allele frequency. We hypothesized that schizophrenia risk genes would be more interactive than random genes and we confirmed this relationship. Gene-gene interactions may cause network ripple effects that spread and amplify small individual effects of risk variants. The modeling revealed that the number of risk alleles required to achieve the threshold for susceptibility will be determined by the average functional locus output (FLO) associated with a risk allele, the risk allele frequency (RAF), the number of protective variants present and the extent of gene interactions within and between risk loci. The model can account for the quantitative impact of protective variants as well as CNVs on disease susceptibility. The fact that non-affected individuals must carry a non-trivial burden of risk alleles suggests that genetic susceptibility will inevitably reach the threshold for schizophrenia at a recurring frequency in the population.

Catalytic Reaction Model of Suicide.

Suicide is a devastating outcome of unresolved issues that affect mental health, general wellbeing and socioeconomic stress. The biology of suicidal behavior is still poorly understood, although progress has been made. Suicidal behavior runs in families and genetic studies have provided initial glimpses into potential genes that contribute to suicide risk. Here, we attempt to unify the biology and behavioral dimensions into a model that can guide research in this area. The proposed model envisions suicidal behavior as a catalytic reaction that may result in suicide depending on the conditions, analogously to enzyme catalysis of chemical reactions. A wide array of substrates or reactants, such as hopelessness, depression, debilitating illnesses and diminished motivation can mobilize suicidal thoughts and behaviors (STBs), which can then catalyze the final step/act of suicide. Here, we focus on three biological substrates in particular: threat assessment, motivation to engage in life and impulsivity. Genetic risk factors can affect each of these processes and tilt the balance toward suicidal behavior when existential crises (real or perceived) emerge such as loss of a loved one, sudden changes in social status or serious health issues. Although suicide is a uniquely human behavior, many of the fundamental biological processes are evolutionarily conserved. Insights from animal models may help to shape our understanding of suicidal behavior in man. By examining counterparts of the major biological processes in other organisms, new ideas about the role of genetic risk factors may emerge along with possible therapeutic interventions or preventive measures.

Protein Receptors Evolved from Homologous Cohesion Modules That Self-Associated and Are Encoded by Interactive Networked Genes.

Previously, it was proposed that protein receptors evolved from self-binding peptides that were encoded by self-interacting gene segments (inverted repeats) widely dispersed in the genome. In addition, self-association of the peptides was thought to be mediated by regions of amino acid sequence similarity. To extend these ideas, special features of receptors have been explored, such as their degree of homology to other proteins, and the arrangement of their genes for clues about their evolutionary origins and dynamics in the genome. As predicted, BLASTP searches for homologous proteins detected a greater number of unique hits for queries with receptor sequences than for sequences of randomly-selected, non-receptor proteins. This suggested that the building blocks (cohesion modules) for receptors were duplicated, dispersed, and maintained in the genome, due to structure/function relationships discussed here. Furthermore, the genes coding for a representative panel of receptors participated in a larger number of gene-gene interactions than for randomly-selected genes. This could conceivably reflect a greater evolutionary conservation of the receptor genes, with their more extensive integration into networks along with inherent properties of the genes themselves. In support of the latter possibility, some receptor genes were located in active areas of adaptive gene relocation/amalgamation to form functional blocks of related genes. It is suggested that adaptive relocation might allow for their joint regulation by common promoters and enhancers, and affect local chromatin structural domains to facilitate or repress gene expression. Speculation is included about the nature of the coordinated communication between receptors and the genes that encode them.

Relapse of Psychosis Status Post Meningioma Resection.

A 53-year-old Caucasian female with a previous psychiatric history of bipolar I disorder and attention deficit hyperactivity disorder presented to the emergency department after endorsing 10-11 months of auditory and visual hallucinations, persecutory delusions, depression, anosmia, weakness of lower extremities, and headache. The patient described her auditory hallucinations as non-commanding voices talking to her about her family, her visual hallucinations as seeing "shadows and shapes," and her paranoid delusions as people coming after her. The patient had sustained a fall a week and a half earlier, requiring eight sutures to her posterior scalp. Her MRI of the brain showed a well-circumscribed 3.5 x 4.7 x 3.2 cm mass in the floor of the anterior cranial fossa. Computer tomography of the brain showed a 4.5 cm mass near the anterior interhemispheric fissure and edema in the right frontal cortex. Meningioma resection resulted in the cessation of hallucinations and delusions for one week. However, the patient was brought back to the emergency department because her auditory hallucinations and delusions returned. This case report demonstrates residual psychosis, even after frontal meningioma resection.

Analysis of Major Depression Risk Genes Reveals Evolutionary Conservation, Shared Phenotypes, and Extensive Genetic Interactions.

Major depressive disorder (MDD) affects around 15% of the population at some stage in their lifetime. It can be gravely disabling and it is associated with increased risk of suicide. Genetics play an important role; however, there are additional environmental contributions to the pathogenesis. A number of possible risk genes that increase liability for developing symptoms of MDD have been identified in genome-wide association studies (GWAS). The goal of this study was to characterize the MDD risk genes with respect to the degree of evolutionary conservation in simpler model organisms such as Caenorhabditis elegans and zebrafish, the phenotypes associated with variation in these genes and the extent of network connectivity. The MDD risk genes showed higher conservation in C. elegans and zebrafish than genome-to-genome comparisons. In addition, there were recurring themes among the phenotypes associated with variation of these risk genes in C. elegans. The phenotype analysis revealed enrichment for essential genes with pleiotropic effects. Moreover, the MDD risk genes participated in more interactions with each other than did randomly-selected genes from similar-sized gene sets. Syntenic blocks of risk genes with common functional activities were also identified. By characterizing evolutionarily-conserved counterparts to the MDD risk genes, we have gained new insights into pathogenetic processes relevant to the emergence of depressive symptoms in man.

Na+ leak-current channel (NALCN) at the junction of motor and neuropsychiatric symptoms in Parkinson's disease.

Parkinson's disease (PD) is a debilitating movement disorder often accompanied by neuropsychiatric symptoms that stem from the loss of dopaminergic function in the basal ganglia and altered neurotransmission more generally. Akinesia, postural instability, tremors and frozen gait constitute the major motor disturbances, whereas neuropsychiatric symptoms include altered circadian rhythms, disordered sleep, depression, psychosis and cognitive impairment. Evidence is emerging that the motor and neuropsychiatric symptoms may share etiologic factors. Calcium/ion channels (CACNA1C, NALCN), synaptic proteins (SYNJ1) and neuronal RNA-binding proteins (RBFOX1) are among the risk genes that are common to PD and various psychiatric disorders. The Na+ leak-current channel (NALCN) is the focus of this review because it has been implicated in dystonia, regulation of movement, cognitive impairment, sleep and circadian rhythms. It regulates the resting membrane potential in neurons, mediates pace-making activity, participates in synaptic vesicle recycling and is functionally co-localized to the endoplasmic reticulum (ER)-several of the major processes adversely affected in PD. Here, we summarize the literature on mechanisms and pathways that connect the motor and neuropsychiatric symptoms of PD with a focus on recurring relationships to the NALCN. It is hoped that the various connections outlined here will stimulate further discussion, suggest additional areas for exploration and ultimately inspire novel treatment strategies.

Candidate risk genes for bipolar disorder are highly conserved during evolution and highly interconnected.

OBJECTIVES: Bipolar disorder (BPD) is a highly heritable psychiatric disorder whose genetic complexity and pathogenetic mechanisms are still being unraveled. The main goal of this work was to characterize BPD risk-gene candidates (identified by Nurnberger et al., JAMA Psychiatry 71:657, 2014, and Stahl et al., Nat. Genet. 51:793, 2019) with respect to their evolutionary conservation, associated phenotypes, and extent of gene-gene interactions. METHODS: Database searches and BLAST were used to identify homologous counterparts of human BPD risk genes in C. elegans, zebrafish, and Drosophila. Phenotypes associated with the C. elegans genes were annotated and searched. With GeneMANIA, we characterized and quantified gene-gene interactions among members of the BPD gene set in comparison to randomly chosen gene sets of the same size. RESULTS: BPD risk genes are highly conserved across species and are enriched for essential genes and genes associated with lethality and altered life span. They are significantly more interactive with each other in comparison to random genes. We identified syntenic blocks of risk genes, which provided potential insights into molecular pathways and co-morbidities associated with BPD including coronary disease, obesity, and decreased life expectancy. CONCLUSIONS: BPD risk genes appear to be special in terms of their degree of conservation, interconnectedness, and pleiotropic effects that extend beyond a role in brain function. Key hub genes or pleiotropic regulatory components may represent attractive targets for future drug discovery.

Clozapine, nimodipine and endosulfan differentially suppress behavioral defects caused by gain-of-function mutations in a two-pore domain K+ channel (UNC-58).

Two-pore domain K+ channels (K2Ps) regulate the resting membrane potential in excitable cells and determine ease of depolarization. Gain-of-function (gf) mutations in one of these channels (unc-58) in C. elegans switch it to a Na+ conductance channel and cause tremors, paralysis and other defects. We hypothesized that it should be possible to identify drugs that corrected these defects in unc-58(gf) mutant animals by blocking or modulating the over-active channels. We examined dispersal of animals on food because the absence of effective forward locomotion is the most obvious defect. In addition, we quantified egg release over 24 h. Starting with a known inhibitor of mammalian K2Ps and directed structure-based screening, we evaluated numerous drugs in these assays. Loratadine, which inhibits human KCNK18, significantly improved movement as did methiothepin. We confirmed that endosulfan, a GABA-A receptor antagonist, corrected locomotion in the unc-58(gf) strains. Based on structural similarities to other hits, we found that clozapine, loxapine and amoxapine potently suppressed abnormal phenotypes. Curiously, nimodipine, a Ca++-channel blocker, dramatically improved movement and egg laying in unc-58(e665), but not unc-58(n495) animals. Molecular modeling provided initial insights into a possible basis for this difference based on the location of the e665 and n495 mutations. This research may lead to identification of novel K2P modulators and potential leads for drug discovery.

Generation of Phenothiazine with Potent Anti-TLK1 Activity for Prostate Cancer Therapy.

Through in vitro kinase assays and docking studies, we report the synthesis and biological evaluation of a phenothiazine analog J54 with potent TLK1 inhibitory activity for prostate cancer (PCa) therapy. Most PCa deaths result from progressive failure in standard androgen deprivation therapy (ADT), leading to metastatic castration-resistant PCa. Treatments that can suppress the conversion to mCRPC have high potential to be rapidly implemented in the clinics. ADT results in increased expression of TLK1B, a key kinase upstream of NEK1 and ATR and mediating the DNA damage response that typically results in temporary cell-cycle arrest of androgen-responsive PCa cells, whereas its abrogation leads to apoptosis. We studied J54 as a potent inhibitor of this axis and as a mediator of apoptosis in vitro and in LNCaP xenografts, which has potential for clinical investigation in combination with ADT. J54 has low affinity for the dopamine receptor in modeling and competition studies and weak detrimental behavioral effects in mice and C. elegans.

Genomic Chaos Begets Psychiatric Disorder.

The processes that created the primordial genome are inextricably linked to current day vulnerability to developing a psychiatric disorder as summarized in this review article. Chaos and dynamic forces including duplication, transposition, and recombination generated the protogenome. To survive early stages of genome evolution, self-organization emerged to curb chaos. Eventually, the human genome evolved through a delicate balance of chaos/instability and organization/stability. However, recombination coldspots, silencing of transposable elements, and other measures to limit chaos also led to retention of variants that increase risk for disease. Moreover, ongoing dynamics in the genome creates various new mutations that determine liability for psychiatric disorders. Homologous recombination, long-range gene regulation, and gene interactions were all guided by spooky action-at-a-distance, which increased variability in the system. A probabilistic system of life was required to deal with a changing environment. This ensured the generation of outliers in the population, which enhanced the probability that some members would survive unfavorable environmental impacts. Some of the outliers produced through this process in man are ill suited to cope with the complex demands of modern life. Genomic chaos and mental distress from the psychological challenges of modern living will inevitably converge to produce psychiatric disorders in man.

Novel pharmacological modulation of dystonic phenotypes caused by a gain-of-function mutation in the Na+ leak-current channel.

The Na leak-current channel (NALCN) regulates the resting membrane potential in excitable cells, thus determining the likelihood of depolarization in response to incoming signals. Gain-of-function (gf) mutations in this channel are associated with severe dystonic movement disorders in man. Currently, there are no known pharmacological antagonists or selective modulators of this important channel. A gain-of-function mutation in NALCN of C. elegans [known as unc-77(e625)] causes uncoordinated, hyperactive locomotion. We hypothesized that this hyperactive phenotype can be rescued with pharmacological modulators. Here, we summarize the results of targeted drug screening aimed at identification of drugs that corrected locomotion deficits in unc-77(e625) animals. To assay hyperactive locomotion, animals were acutely removed from food and characteristic foraging movements were quantified. Drug screening revealed that 2-aminoethoxydiphenyl borate (2-ABP), nifedipine, nimodipine, flunarizine and ethoxzolamide significantly decreased abnormal movements in unc-77(e625) animals. 2-APB also corrected egg release and coiling deficits in this strain. In addition, serotonin and dopamine both reduced hyperactive locomotion, consistent with regulatory interactions between these systems and the NALCN. 2-APB induced movement phenotypes in wild-type animals that faithfully mimicked those observed in NALCN knockout strains, which suggested that this drug may directly block the channel. Moreover, 2-APB and flunarizine showed significant structural similarities suggestive of overlap in their mode of action. Together, these studies have revealed new insights into regulation of NALCN function and led to the discovery of a potential pharmacological antagonist of the NALCN.

Dosage sensitivity intolerance of VIPR2 microduplication is disease causative to manifest schizophrenia-like phenotypes in a novel BAC transgenic mouse model.

Recent genome-wide association studies (GWAS) have identified copy number variations (CNVs) at chromosomal locus 7q36.3 that significantly contribute to the risk of schizophrenia, with all of the microduplications occurring within a single gene: vasoactive intestinal peptide receptor 2 (VIPR2). To confirm disease causality and translate such a genetic vulnerability into mechanistic and pathophysiological insights, we have developed a series of conditional VIPR2 bacterial artificial chromosome (BAC) transgenic mouse models of VIPR2 CNV. VIPR2 CNV mouse model recapitulates gene expression and signaling deficits seen in human CNV carriers. VIPR2 microduplication in mice elicits prominent dorsal striatal dopamine dysfunction, cognitive, sensorimotor gating, and social behavioral deficits preceded by an increase of striatal cAMP/PKA signaling and the disrupted early postnatal striatal development. Genetic removal of VIPR2 transgene expression via crossing with Drd1a-Cre BAC transgenic mice rescued the dopamine D2 receptor abnormality and multiple behavioral deficits, implicating a pathogenic role of VIPR2 overexpression in dopaminoceptive neurons. Thus, our results provide further evidence to support the GWAS studies that the dosage sensitivity intolerance of VIPR2 is disease causative to manifest schizophrenia-like dopamine, cognitive, and social behavioral deficits in mice. The conditional BAC transgenesis offers a novel strategy to model CNVs with a gain-of -copies and facilitate the genetic dissection of when/where/how the genetic vulnerabilities affect development, structure, and function of neural circuits. Our findings have important implications for therapeutic development, and the etiology-relevant mouse model provides a useful preclinical platform for drug discovery.

Surprising conservation of schizophrenia risk genes in lower organisms reflects their essential function and the evolution of genetic liability.

Schizophrenia is a devastating psychiatric illness that affects approximately 1% of the population. Genetic variation in multiple genes causes elevated risk for the disorder, but the molecular basis is inadequately understood and it is not clear how risk genes have evolved and persisted in the genome. To address these issues, we have identified orthologs/homologs of 344 schizophrenia risk genes (from the Psychiatric Genomics Consortium dataset) in lower organisms, including C. elegans, Drosophila and zebrafish, along with phenotypes produced by genetic disruption in C. elegans. Schizophrenia risk genes were evolutionarily conserved at significantly higher rates in C. elegans (81%) and zebrafish (88%) than genes in general for these two species (40-70%). The risk-gene equivalents were highly (~3-fold) enriched for essential genes consistent with polygenic mutation threshold models, which propose that genetic susceptibility results from the inevitable expression of harmful combinations of risk variants in the population. Most notably, numerous examples of cross-species synteny revealed how blocks of risk genes geared toward a shared biological purpose coalesced into proximity during evolution. We obtained initial evidence that schizophrenia risk genes affected different stages of development, potentially allowing differential modulation by the environment. Taken together, studies of the conservation of schizophrenia risk genes in simple model organisms provided novel insights into the molecular basis for genetic susceptibility to a complex human psychiatric disorder.

Crossing the Worm-Brain Barrier by Using Caenorhabditis elegans to Explore Fundamentals of Human Psychiatric Illness.

Endophenotypes and Research Domain Criteria (RDoC) represent recent efforts to deconvolute psychiatric illnesses into fundamental symptom clusters or biological markers more closely linked to genetic influences. By taking this one step farther, these biomarkers can be reduced to protophenotypes - endophenotypes conserved during evolution - with counterparts in lower organisms including Caenorhabditis elegans and Drosophila. Striking conservation in C. elegans of genes that increase the risk for mental illness bolsters the relevance of this model system for psychiatric research. Here, I review the characterization of several protophenotypes that are relevant for asociality, avolition/anhedonia, prepulse inhibition, and anorexia. Interestingly, the analogous behavioral defects in C. elegans are also corrected by psychotropic drugs used to treat the corresponding symptoms in man and/or are mediated by the same neurotransmitters. Overall, there is much we can learn about the complex human brain by studying simpler nervous systems directing evolutionarily conserved behaviors. The potential for generating important new insights from model organisms appears limitless when we begin to recognize the vestiges of evolution in ourselves.

Insulin Signaling Deficiency Produces Immobility in Caenorhabditis elegans That Models Diminished Motivation States in Man and Responds to Antidepressants.

Defects in insulin signaling have been reported in schizophrenia and major depressive disorder, which also share certain negative symptoms such as avolition, anhedonia, and apathy. These symptoms reflect diminished motivational states, which have been modeled in rodents as increased immobility in the forced swimming test. We have discovered that loss-of-function mutations in the insulin receptor (daf-2) and syntaxin (unc-64) genes in Caenorhabditis elegans, brief food deprivation, and exposure to DMSO produce immobility and avolition in non-dauer adults. The animals remain responsive to external stimuli; however, they fail to forage and will remain in place for >12 days or until they die. Their immobility can be prevented with drugs used to treat depression and schizophrenia and that reduce immobility in the forced swimming test. This includes amitriptyline, amoxapine, clozapine, and olanzapine, but not benzodiazepines and haloperidol. Recovery experiments confirm that immobility is induced and maintained by excessive signaling via serotonergic and muscarinic cholinergic pathways. The immobility response described here represents a potential protophenotype for avolition/anhedonia in man. This work may provide clues about why there is a significant increase in depression in patients with diabetes and suggest new therapeutic pathways for disorders featuring diminished motivation as a prominent symptom.

Two adjacent phenylalanines in the NMDA receptor GluN2A subunit M3 domain interactively regulate alcohol sensitivity and ion channel gating.

The N-methyl-d-aspartate (NMDA) receptor is a key target of ethanol action in the central nervous system. Alcohol inhibition of NMDA receptor function involves small clusters of residues in the third and fourth membrane-associated (M) domains. Previous results from this laboratory have shown that two adjacent positions in the M3 domain, F636 and F637, can powerfully regulate alcohol sensitivity and ion channel gating. In this study, we report that these positions interact with one another in the regulation of both NMDA receptor gating and alcohol action. Using dual mutant cycle analysis, we detected interactions among various substitution mutants at these positions with respect to regulation of glutamate EC50, steady-state to peak current ratios (Iss:Ip), mean open time, and ethanol IC50. This interaction apparently involves a balancing of forces on the M3 helix, such that the disruption of function due to a substitution at one position can be reversed by a similar substitution at the other position. For example, tryptophan substitution at F636 or F637 increased or decreased channel mean open time, respectively, but tryptophan substitution at both positions did not alter open time. Interestingly, the effects of a number of mutations on receptor kinetics and ethanol sensitivity appeared to depend upon subtle structural differences, such as those between the isomeric amino acids leucine and isoleucine, as they could not be explained on the basis of sidechain molecular volume or hydrophilicity.

Akinesia and freezing caused by Na+ leak-current channel (NALCN) deficiency corrected by pharmacological inhibition of K+ channels and gap junctions.

The Na+ leak-current channel (NALCN) regulates locomotion, respiration, and intellectual development. Previous work highlighted striking similarities between characteristic movement phenotypes of NALCN-deficient animals (Drosophila and Caenorhabditis elegans) and the major symptoms of Parkinson's disease and primary progressive freezing gait. We have discovered novel physiological connections between the NALCN, K+ channels, and gap junctions that mediate regulation of locomotion in C. elegans. Drugs that block K+ channels and gap junctions or that activate Ca++ channels significantly improve movement of NALCN-deficient animals. Loss-of-function of the NALCN creates an imbalance in ions, including K+ and Ca++ , that interferes with normal cycles of depolarization-repolarization. This work suggests new therapeutic strategies for certain human movement disorders. J. Comp. Neurol. 525:1109-1121, 2017. © 2016 Wiley Periodicals, Inc.

Different sites of alcohol action in the NMDA receptor GluN2A and GluN2B subunits.

The NMDA receptor is a major target of alcohol action in the CNS, and recent behavioral and cellular studies have pointed to the importance of the GluN2B subunit in alcohol action. We and others have previously characterized four amino acid positions in the third and fourth membrane-associated (M) domains of the NMDA receptor GluN2A subunit that influence both ion channel gating and alcohol sensitivity. In this study, we found that substitution mutations at two of the four corresponding positions in the GluN2B subunit, F637 and G826, influence ethanol sensitivity and ion channel gating. Because position 826 contains a glycine residue in the native protein, we focused our attention on GluN2B(F637). Substitution mutations at GluN2B(F637) significantly altered ethanol IC50 values, glutamate EC50 values for peak (Ip) and steady-state (Iss) current, and steady-state to peak current ratios (Iss:Ip). Changes in apparent glutamate affinity were not due to agonist trapping in desensitized states, as glutamate Iss EC50 values were not correlated with Iss:Ip values. Ethanol sensitivity was correlated with values of both Ip and Iss glutamate EC50, but not with Iss:Ip. Values of ethanol IC50, glutamate EC50, and Iss:Ip for mutants at GluN2B(F637) were highly correlated with the corresponding values for mutants at GluN2A(F636), consistent with similar functional roles of this position in both subunits. These results demonstrate that GluN2B(Phe637) regulates ethanol action and ion channel function of NMDA receptors. However, despite highly conserved M domain sequences, ethanol's actions on GluN2A and GluN2B subunits differ.