Analyzing the Effects of Benzodiazepines on the Structure and Phosphorylation of Type A γ-Aminobutyric Acid Receptors

Benzodiazepines are potent anxiolytic, anticonvulsant, hypnotic and sedative agents that are among the most widely used drugs to treat both psychiatric and neurological disorders. However, their long-term use results in tolerance and addiction. Accordingly, benzodiazepines are amongst the most highly abused class of prescription drugs. Benzodiazepines exert their behavioral effects via allosteric potentiation of γ-aminobutyric acid (GABA) type A receptors, the principal mediators of synaptic and tonic inhibition in the brain. GABA type A receptors are also the molecular targets for barbiturates, another widely abused class of hypnotic and sedative drugs, further highlighting their roles in drug addiction.

Structurally GABA type A receptors are heteropentamers that can be assembled from 7 subunit classes; α1-6, β1-3, γ1-3, δ, ε, θ, and π-subunits, providing the molecular basis for extensive receptor structural heterogeneity throughout the brain. Consensus opinion suggests that synaptic benzodiazepine GABA type A receptor subtypes are arranged from α1-3, β1-3 and γ2 subunits, and the activation of subtypes containing α1 and/or α2 subunits play essential roles in mediating their rewarding effects on behavior. In this proposal, we will assess if persistent exposure of mice to benzodiazepines induces sustained effects on the composition of these distinct receptor subtypes, their association with components of the inhibitory proteome, or modifies their phosphorylation. To perform these experiments, we will make use of recently developed knock-in mice in which distinct fluorescent proteins and reporter epitopes have been inserted between amino acids 4 and 5 within the N-terminus of the mature α1 and α2 subunits; mKate-α1 and pHluorin-α2, respectively. Importantly these modifications are functionally silent and do not compromise the formation of inhibitory synapses, neuronal architecture, or animal behavior (Nakamura et al., 2016). However, these additions facilitate the isolation of native multiple protein complexes containing individual GABA type A receptors subtypes. They will be used here in combination with quantitative proteomics to assess if benzodiazepine exposure modifies the composition of α1 and α2 subunit-containing GABA type A receptors in addition to their phosphorylation status.

Since joining the center in 2019 we have made progress on deciphering the structurally heterogeneity of GABA_AR subunits in the brain as described in two recent publications (Hines et al., 2018; Nathanson et al., 2019).

Literature Cited

Hines, R.M., Maric, H.M., Hines, D.J., Modgil, A., Panzenelli, P., Nakamura, Y., Nathanson, A., Cross, A., Brandon, N., Davies, P., Fritschy, J.M., Schindelin, H., and Moss S.J. (2018). Decreasing the interaction of the GABAAR α2 subunit with collybistin results in spontaneous seizures and premature death. Nat Comm 9(1):3130.

Nakamura, Y., Morrow, D.H., Modgil, A., Huyghe, D., Deeb, T.Z., Lumb, M.J., Davies, P.A. and Moss, S.J. (2016). Proteomic characterization of inhibitory synapses using a novel pHluorin-tagged GABA type A receptor α2 subunit knockin mouse. J. Biol. Chem. 291(23):12394-407.

Nathanson, A.J., Zhang, Y., Smalley, J.S. Thomas A. Ollerhead, O.A., Rodriguez Santos, M.A. Peter M. Andrews, P.M., Bope, C., Brandon, N.J., Hines, R.M., Davies, P.A. and Moss S.J. (2019). Identification of a core amino acid motif within the α subunit of GABAARs that promotes inhibitory synaptogenesis and resilience to seizures. Cell Reports 28(3):670-681.e8. doi: 10.1016/j.celrep.2019