Type A γ-aminobutyric acid receptors (GABAARs) control neuronal excitability1. They are targets for the treatment of neurological diseases and disorders and also for general anesthetics. The underlying mechanisms of these drugs’ action on GABAARs remain to be determined.
One of the mechanisms is to potentiate function of GABAARs via binding to the transmembrane domain (TMD)2. Ample experimental evidence suggests that the TMD of GABAARs harbors sites for the primary actions of general anesthetics and neurosteroids. The TMD plays an essential role in functional transitions among the resting, activated, and desensitized states of these Cl－-conducting channels.
Alphaxalone (5α-pregnan-3α-ol-11,20 dione) is a potent neurosteroid anesthetic. The anxiolytic, anticonvulsant, analgesic, and sedative-hypnotic effects of alphaxalone have been linked to its potentiation of GABA-evoked currents and direct activation of GABAARs3. However, the data about the alphaxalone binding site in GABAARs and the underlying structural basis of alphaxalone’s action are sparse.
Figure: Alphaxalone-induced structural changes at the bottom of the TMD (a) Bottom view of overlaid TM1-TM2 structures of the apo (orange) and alphaxalone-bound (cyan) α1GABAAR chimera. (b) Side view of overlaid structures of apo (principal subunit – gold; complementary subunit – orange) and alphaxalone-bound (principal subunit – blue; complementary subunit – cyan) α1GABAAR chimera. For clarity, only TM2 and TM3 are shown in the principal subunit and only TM1 and TM2 are shown in the complementary subunit. The arrow highlights structural perturbations originating from the alphaxalone binding site near W246 through the TM1-TM2 linker to the pore-lining residues P253 (-2′) and V257 (2′). (c) The 2FO-FC electron density maps (blue mesh, contoured at 1 σ) covering TM1-TM2 in the apo (left) and alphaxalone-bound (right) α1GABAAR chimera. The sidechains are shown only for residues W246 to V257 (2′).
Synergetic combination of different imaging and spectroscopic synchrotron techniques performed in ALBA and APS (USA) has discovered new aspects about astrocytes cells of this neurodegenerative disease.
Results, published in Analytical Chemistry, show significant differences between ALS and control astrocytes, including structural, chemical and macromolecular anomalies. Amyotrophic lateral sclerosis (ALS) is a fatal progressive neurodegenerative disease that causes the degeneration and death of neurons that control voluntary muscles. Still today the causes of this disease are unknown in 90% of the cases. However, some of them are caused by the mutation of sod1 gene. This gene encodes an enzyme (SOD1) that is involved in cellular protection against oxidative stress. Mutations dramatically alter the biochemical properties of SOD1, in particular its metal binding affinity and its anti-oxidative activity levels. But it is still unknown how these mutations block the normal cell function and lead to death of motor neurons. The ALBA Synchrotron, in collaboration with researchers from the University of Belgrade Pavle Andjus and Stefan Stamenković (who accomplished his PhD thesis using these results) and Vladan Lučić from Max Planck Institute of Biochemistry (Germany), has studied with synchrotron light techniques and classical biochemical laboratory approaches the cellular structural and biochemical changes of this gene mutation in a transgenic animal model of ALS. In particular, scientists have analysed astrocytes, one kind of brain cells that are key players in pathological processes of this disease.
Image: Researcher Tanja Dučić during the experiment performed at ALBA, at the MIRAS beamline.
Study contributes to the understanding of mechanisms involved in neurodevelopmental disorders
Once a disease-related protein or enzyme is identified as a therapeutic target, the study of its three-dimensional structure – the positions of each of its atoms and their interactions – allows a deeper understanding of its mechanisms of action.
This is possible not only for these substances produced by microorganisms, such as viruses or bacteria, capable of attacking our body. It is also possible, for example, to understand molecules normally produced by the human body itself, but which had their structure and function altered due to some genetic mutation.
Thus, in an article recently published in Nature Chemical Biology, Juliana F. de Oliveira, of the Brazilian Biosciences National Laboratory (LNBio), and collaborators elucidates the mechanism of action of a new genetic mutation in the UBE2A gene identified in patients with intellectual disability.
The UBE2A gene is located on the X chromosome and encodes the protein of the same name that participates in the process of “labeling” defective proteins inside the cell. This labeling is done by adding and protein called ubiquitin to the defective proteins as if it were a label. Next, under normal conditions, the defective proteins are sent for degradation.
Image: Overlap of the patient’s UBE2A protein structure (blue) with the normal protein (gray) evidences similarity between them. On the right, it is shown in detail the only altered amino acid in the patient’s protein due to the genetic mutation.
“It was a fantastic experience”, Jette Sandholm Kastrup.
On June 30, 2017 Professor Jette Sandholm Kastrup, University of Copenhagen was granted two shifts of beamtime at BioMAX by the Program Advisory Committee (PAC) and the MAX IV Laboratory Management for the project “Molecular recognition of agonists, antagonists and positive allosteric modulators at ionotropic glutamate receptors”.
The ionotropic glutamate receptors (iGluRs) are highly abundant in the central nervous system (CNS) and mediate fast synaptic neurotransmission. Dysfunction of the glutamatergic system has been associated with various diseases in the CNS, e.g. depression, Parkinson’s and Alzheimer’s diseases and epilepsy. The iGluRs are for example considered an attractive and appropriate target for the discovery of cognitive enhancers.