Emerging Roles of γ Aminobutyric Acid (GABA) Gated Channels in Plant Stress Tolerance

The signaling role for γ-Aminobutyric acid (GABA) has been documented in animals for over seven decades. However, a signaling role for GABA in plants is just beginning to emerge with the discovery of putative GABA binding site/s and GABA regulation of anion channels. In this review, we explore the role of GABA in plant growth and development under abiotic stress, its interactions with other signaling molecules and the probability that there are other anion channels with important roles in stress tolerance that are gated by GABA.

The desensitization pathway of GABAA receptors, one subunit at a time

GABAA receptors mediate most inhibitory synaptic transmission in the brain of vertebrates. Following GABA binding and fast activation, these receptors undergo a slower desensitization, the conformational pathway of which remains largely elusive. To explore the mechanism of desensitization, we used concatemeric α1β2γ2 GABAA receptors to selectively introduce gain-of-desensitization mutations one subunit at a time. A library of twenty-six mutant combinations was generated and their bi-exponential macroscopic desensitization rates measured. Introducing mutations at the different subunits shows a strongly asymmetric pattern with a key contribution of the γ2 subunit, and combining mutations results in marked synergistic effects indicating a non-concerted mechanism. Kinetic modelling indeed suggests a pathway where subunits move independently, the desensitization of two subunits being required to occlude the pore. Our work thus hints towards a very diverse and labile conformational landscape during desensitization, with potential implications in physiology and pharmacology.

The desensitization pathway of GABAA receptors, one subunit at a time

GABAA receptors mediate most inhibitory synaptic transmission in the brain of vertebrates. Following GABA binding and fast activation, these receptors undergo a slower desensitization, whose conformational pathway remains largely elusive. To explore the mechanism of desensitization, we used concatemeric α1β2γ2 GABAA receptors to selectively introduce gain-of-desensitization mutations one subunit at a time. A library of twenty-six mutant combinations was generated and their bi-exponential macroscopic desensitization rates measured. Introducing mutations at the different subunits shows a strongly asymmetric pattern with a key contribution of the γ2 subunit, and combining mutations results in marked synergistic effects indicating a non-concerted mechanism. Kinetic modelling indeed suggests a pathway where subunits move independently, the desensitization of two subunits being required to occlude the pore. Our work thus hints towards a very diverse and labile conformational landscape during desensitization, with potential implications in physiology and pharmacology.

Conformational transitions induced by γ-amino butyrate binding in GabR, a bacterial transcriptional regulator

GabR from Bacillus subtilis is a transcriptional regulator of the MocR subfamily of GntR regulators. The MocR architecture is characterized by the presence of an N-terminal winged-Helix-Turn-Helix domain and a C-terminal domain folded as the pyridoxal 5′-phosphate (PLP) dependent aspartate aminotransferase (AAT). The two domains are linked by a peptide bridge. GabR activates transcription of genes involved in γ-amino butyrate (GABA) degradation upon binding of PLP and GABA. This work is aimed at contributing to the understanding of the molecular mechanism underlying the GabR transcription activation upon GABA binding. To this purpose, the structure of the entire GabR dimer with GABA external aldimine (holo-GABA) has been reconstructed using available crystallographic data. The structure of the apo (without any ligand) and holo (with PLP) GabR forms have been derived from the holo-GABA. An extensive 1 μs comparative molecular dynamics (MD) has been applied to the three forms. Results showed that the presence of GABA external aldimine stiffens the GabR, stabilizes the AAT domain in the closed form and couples the AAT and HTH domains dynamics. Apo and holo GabR appear more flexible especially at the level of the HTH and linker portions and small AAT subdomain.

Altered inhibitory synapses in de novo GABRA5 and GABRA1 mutations associated with early onset epileptic encephalopathies

We performed next generation sequencing on 1696 patients with epilepsy and intellectual disability using a gene panel with 480 epilepsy-related genes including all GABAA receptor subunit genes (GABRs), and we identified six de novo GABR mutations, two novel GABRA5 mutations (c.880G>T, p.V294F and c.1238C>T, p.S413F), two novel GABRA1 mutations (c.778C>T, p.P260S and c.887T>C, p.L296S/c.944G>T, p.W315L) and two known GABRA1 mutations (c.335G>A, p.R112Q and c.343A>G, p.N115D) in six patients with intractable early onset epileptic encephalopathy. The α5(V294F and S413F) and α1(P260S and L296S/W315L) subunit residue substitutions were all in transmembrane domains, while the α1(R112Q and N115R) subunit residue substitutions were in the N-terminal GABA binding domain. Using multidisciplinary approaches, we compared effects of mutant GABAA receptor α5 and α1 subunits on the properties of recombinant α5β3γ2 and α1β3γ2 GABAA receptors in both neuronal and non-neuronal cells and characterized their effects on receptor clustering, biogenesis and channel function. GABAA receptors containing mutant α5 and α1 subunits all had reduced cell surface and total cell expression with altered endoplasmic reticulum processing, impaired synaptic clustering, reduced GABAA receptor function and decreased GABA binding potency. Our study identified GABRA5 as a causative gene for early onset epileptic encephalopathy and expands the mutant GABRA1 phenotypic spectrum, supporting growing evidence that defects in GABAergic neurotransmission contribute to early onset epileptic encephalopathy phenotypes.