Interaction of betaine with the γ-aminobutyric acid (GABA) transporters

Betaine, N-trimethyl glycine, is a natural, stable, and non-toxic osmolyte, whose supplementation is reported to ameliorate symptoms in neurological and neurodegenerative diseases. Evidence of positive action is reported in Alzheimer’s, Parkinson’s disease, and schizophrenia, despite that there is uncertainty around its transport and action mechanism in the brain. Although there are betaine transporters present in the brain i.e., betaine/γ-aminobutyric acid (GABA) transporter 1 (SLC6A12) and the sodium-coupled neutral amino acid transporter 2 (SLC38A2), their low expression propose that the positive effect of betaine should be related to interaction with proteins of the GABAergic pathways. By two-electrode voltage clamp experiments in Xenopus laevis oocytes heterologously expressing rGAT1, we demonstrate that betaine induces dose-, voltage-, and Na+ dependent inward transport currents, that are blocked by GAT1 specific inhibitors. The affinity of betaine for GAT1 (K0.5,betaine≈11mM at -60mV) is greatly lower than for GABA, though coherent with its physiological concentration in neuronal tissue. The transport of betaine by GAT1 has also been confirmed using the radiolabelled release assay in HEK293 cells, LCMS-MS technique, and supported by molecular docking. A peculiar behaviour of GAT1 was revealed by investigating GABA and betaine relationship. Betaine plays a dual role in GAT1 transport: at μM concentration it blocks the GABA transport, given the extracellular GABA is lower than its K0.5,GABA (≈16 μM at -60mV), at mM concentration it behaves like a secondary substrate. Analysing the cytoplasmic presence of GABA and betaine by LCMS-MS in oocytes expressing rGAT1 incubated in different GABA and betaine concentration, confirmed the electrophysiological findings. Our results show that betaine, by preventing the uptake of GABA, can increase the strength of the inhibitory pathway when GABA extracellular levels are low. This modulatory behaviour supports a possible role in maintaining excitatory/inhibitory balance in the central nervous system and the positive effect on neurological conditions.