Molecular and Clinical Repercussions of GABA Transporter 1 Variants Gone Amiss: Links to Epilepsy and Developmental Spectrum Disorders

Abstract

The human γ-aminobutyric acid (GABA) transporter 1 (hGAT-1) is the first member of the solute carrier 6 (SLC6) protein superfamily. GAT-1 (SLC6A1) is one of the main GABA transporters in the central nervous system. Its principal physiological role is retrieving GABA from the synapse into neurons and astrocytes, thus swiftly terminating neurotransmission. GABA is a key inhibitory neurotransmitter and shifts in GABAergic signaling can lead to pathological conditions, from anxiety and epileptic seizures to schizophrenia. Point mutations in the SLC6A1 gene frequently give rise to epilepsy, intellectual disability or autism spectrum disorders in the afflicted individuals. The mechanistic routes underlying these are still fairly unclear. Some loss-of-function variants impair the folding and intracellular trafficking of the protein (thus retaining the transporter in the endoplasmic reticulum compartment), whereas others, despite managing to reach their bona fide site of action at the cell surface, nonetheless abolish GABA transport activity (plausibly owing to structural/conformational defects). Whatever the molecular culprit(s), the physiological aftermath transpires into the absence of functional transporters, which in turn perturbs GABAergic actions. Dozens of mutations in the kin SLC6 family members are known to exhort protein misfolding. Such events typically elicit severe ailments in people, e.g., infantile parkinsonism-dystonia or X-linked intellectual disability, in the case of dopamine and creatine transporters, respectively. Flaws in protein folding can be rectified by small molecules known as pharmacological and/or chemical chaperones. The search for such apt remedies calls for a systematic investigation and categorization of the numerous disease-linked variants, by biochemical and pharmacological means in vitro (in cell lines and primary neuronal cultures) and in vivo (in animal models). We here give special emphasis to the utilization of the fruit fly Drosophila melanogaster as a versatile model in GAT-1-related studies. Jointly, these approaches can portray indispensable insights into the molecular factors underlying epilepsy, and ultimately pave the way for contriving efficacious therapeutic options for patients harboring pathogenic mutations in hGAT-1.

Keywords: Drosophila melanogaster; GABA transporter 1; autism; epilepsy; gamma-aminobutyric acid (GABA); intellectual disability; protein folding; transporter disease variants.

FIGURE 1

Epilepsy-associated variants mapped onto a human GAT-1 topology. Pathogenic point mutations in the SLC6A1 gene reported in the literature to date are depicted as purple circles on a topology diagram of the human GAT-1. The mutations occur throughout hGAT-1, from transmembrane (TM) domains, to cytoplasmic amino- and carboxyl-termini, as well as intra- and extracellular loop regions. Recurrent mutations are indicated in the magenta font. Frameshift and termination codon mutations are indicated with “fs” and “*”, respectively. Pathogenic mutations found at equivalent conserved residues in other SLC6 transporters are underlined.

FIGURE 2

The putative repercussions of hGAT-1 variants at the GABAergic synapse. A simplified schematic showing the GABAergic synapse in a regular physiological state (A) and in a hGAT-1-triggered epilepsy setting (B). The GABAergic homeostasis is tightly regulated by the neuronal and glial GABA transporters. The absence of plasmalemmal hGAT-1 affects the extracellular clearance of GABA, which results in increased extrasynaptic GABA levels and reduced presynaptic GABA pools affecting the subsequent phasic neurotransmission. The higher levels of extrasynaptic GABA act on the extrasynaptic GABA_A and GABA_B receptors, inducing tonic inhibition.

FIGURE 3

A schematic illustrating the use of fruit flies in studying epilepsy. A cartoon depicting stereotypical behavior observed in Drosophila melanogaster, subjected to mechanical stress. Various stressors such as mechanical, chemical, electrical and heat shock are typically used to induce seizure-like activity in fruit flies. They show varying degrees of sensibility to seizure and duration thereof, depending on the genetic susceptibility. Vortexing flies for a brief 10 s period induces initial seizure-like activity characterized by leg twitches, abdominal contractions, proboscis extensions and wing flapping. The initial phase is followed by the paralysis phase and a subsequent recovery phase, whereby the fly tries to regain its posture. The time required by the fly to fully regain its posture (i.e., enter the recovery phase) is collected for data analysis.