Phenotypic effects from the expression of a deregulated AtGAD1 transgene and GABA pathway suppression mutants in maize

Glutamate decarboxylase (GAD; EC 4.1.1.15) catalyzes the irreversible decarboxylation of glutamate to produce γ-aminobutyric acid (GABA); a ubiquitous non-protein amino acid involved in the regulation of several aspects of plant metabolism and physiology. To study the function of GAD and GABA in maize, we have; 1) introduced native and deregulated forms of AtGAD1 into maize with the intent of increasing the synthesis of GABA and 2) introduced constructs into maize designed to suppress the activity of several GABA shunt, GABA transport and GABA pathway genes. Maize plants expressing the deregulated AtGAD1 exhibit a severe chlorosis and retarded growth phenotype and have high levels of GABA, and Ca++/CaM-independent GAD activity. Plants expressing the suppression constructs for GABA biosynthetic and transport pathway genes had no observable phenotype whereas a knockout of GABA catabolic pathway genes led to growth and developmental defects under standard growth conditions. The implications of this study to our understanding of the action and function of GABA and GAD in crops are discussed.

Association of SLC32A1 Missense Variants With Genetic Epilepsy With Febrile Seizures Plus

ObjectiveTo identify the causative gene in a large unsolved family with genetic epilepsy with febrile seizures plus (GEFS+), we sequenced the genomes of family members, and then determined the contribution of the identified gene to the pathogenicity of epilepsies by examining sequencing data from 2,772 additional patients.MethodsWe performed whole genome sequencing of 3 members of a GEFS+ family. Subsequently, whole exome sequencing (ES) data from 1,165 epilepsy patients from the Epi4K dataset and 1,329 Australian epilepsy patients from the Epi25 dataset was interrogated. Targeted resequencing was performed on 278 patients with FS or GEFS+ phenotypes. Variants were validated and familial segregation examined by Sanger sequencing.ResultsEight previously unreported missense variants were identified in SLC32A1, coding for the vesicular inhibitory amino acid co-transporter VGAT. Two variants co-segregated with the phenotype in 2 large GEFS+ families containing 8 and 10 affected individuals, respectively. Six further variants were identified in smaller families with GEFS+ or idiopathic generalized epilepsy (IGE).ConclusionMissense variants in SLC32A1 cause GEFS+ and IGE. These variants are predicted to alter GABA transport into synaptic vesicles, leading to altered neuronal inhibition. Examination of further epilepsy cohorts will determine the full genotype-phenotype spectrum associated with SLC32A1 variants.

Influence of glutamate and GABA transport on brain excitatory/inhibitory balance

An optimally functional brain requires both excitatory and inhibitory inputs that are regulated and balanced. A perturbation in the excitatory/inhibitory balance—as is the case in some neurological disorders/diseases (e.g. traumatic brain injury Alzheimer’s disease, stroke, epilepsy and substance abuse) and disorders of development (e.g. schizophrenia, Rhett syndrome and autism spectrum disorder)—leads to dysfunctional signaling, which can result in impaired cognitive and motor function, if not frank neuronal injury. At the cellular level, transmission of glutamate and GABA, the principle excitatory and inhibitory neurotransmitters in the central nervous system control excitatory/inhibitory balance. Herein, we review the synthesis, release, and signaling of GABA and glutamate followed by a focused discussion on the importance of their transport systems to the maintenance of excitatory/inhibitory balance.

GRID-independent molecular descriptor analysis and molecular docking studies to mimic the binding hypothesis of γ-aminobutyric acid transporter 1 (GAT1) inhibitors

BackgroundThe γ-aminobutyric acid (GABA) transporter GAT1 is involved in GABA transport across the biological membrane in and out of the synaptic cleft. The efficiency of this Na+coupled GABA transport is regulated by an electrochemical gradient, which is directed inward under normal conditions. However, in certain pathophysiological situations, including strong depolarization or an imbalance in ion homeostasis, the GABA influx into the cytoplasm is increased by re-uptake transport mechanism. This mechanism may lead to extra removal of extracellular GABA which results in numerous neurological disorders such as epilepsy. Thus, small molecule inhibitors of GABA re-uptake may enhance GABA activity at the synaptic clefts.MethodsIn the present study, various GRID-independent molecular descriptor (GRIND) models have been developed to shed light on the 3D structural features of human GAT1 (hGAT1) inhibitors using nipecotic acid and N-diarylalkenyl piperidine analogs. Further, a binding hypothesis has been developed for the selected GAT1 antagonists by molecular docking inside the binding cavity of hGAT1 homology model.ResultsOur results indicate that two hydrogen bond acceptors, one hydrogen bond donor and one hydrophobic region at certain distances from each other play an important role in achieving high inhibitory potency against hGAT1. Our docking results elucidate the importance of the COOH group in hGAT1 antagonists by considering substitution of the COOH group with an isoxazol ring in compound37, which subsequently leads to a three order of magnitude decrease in biological activity of37(IC50= 38 µM) as compared to compound1(IC50= 0.040 µM).DiscussionOur docking results are strengthened by the structure activity relationship of the data series as well as by GRIND models, thus providing a significant structural basis for understanding the binding of antagonists, which may be useful for guiding the design of hGAT1 inhibitors.