Picrotoxin Delineates Different Transport Configurations for Malate and γ Aminobutyric Acid through TaALMT1.

Abstract

Simple SummaryGamma aminobutyric acid (GABA) a non-protein amino acid is an archaic signalling molecule for cellular communication across all kingdoms of life with distinct signalling roles in animals. Emerging research establishes GABA as a signalling molecule in planta. GABA modulates numerous developmental processes and negatively regulates root growth, stomatal aperture, and anion flux under abiotic stress. A putative GABA binding site with homology to the mammalian GABA binding site has been identified in a family of proteins (Aluminium Activated Malate Transporters -ALMT) involved in mediating anion flux and conferring plant aluminium tolerance. Studies have shown that ALMTs are regulated by GABA and transport both anions and GABA. To gain further insights into the mechanism of GABA regulation, it is essential to differentiate between the two transport modes. Pharmacological agents used to characterise mammalian GABA receptors have proved useful in gaining insights into GABA regulation of plant processes. In this study, picrotoxin an inhibitor of anion flux in mammalian GABA receptors was used to investigate the pathways for anion and GABA transport in ALMTs. Results suggest that picrotoxin inhibits anion flux but not GABA flux and can be used to gain further insights into mechanism of GABA regulation of plant proteins.Plant-derived pharmacological agents have been used extensively to dissect the structure–function relationships of mammalian GABA receptors and ion channels. Picrotoxin is a non-competitive antagonist of mammalian GABAA receptors. Here, we report that picrotoxin inhibits the anion (malate) efflux mediated by wheat (Triticum aestivum) ALMT1 but has no effect on GABA transport. The EC50 for inhibition was 0.14 nM and 0.18 nM when the ALMTs were expressed in tobacco BY2 cells and in Xenopus oocytes, respectively. Patch clamping of the oocyte plasma membrane expressing wheat ALMT1 showed that picrotoxin inhibited malate currents from both sides of the membrane. These results demonstrate that picrotoxin inhibits anion efflux effectively and can be used as a new inhibitor to study the ion fluxes mediated by ALMT proteins that allow either GABA or anion transport.