Faculty Bibliography

Gamma-aminobutyric acid (GABA) type A receptors are heteropentameric ligand-gated Cl--channels that mediate the majority of fast inhibitory neurotransmission in the brain. GABAA receptor clustering at postsynaptic sites is critical for the function of inhibitory synapse. Moreover, changes in synaptic receptor concentration are believed to contribute to functional plasticity of neurons. Typical postsynaptic GABAA receptor subtypes are composed of alpha;, beta;, and gamma;2 subunits and they are co-localized at synapses with the putative clustering protein gephyrin, which is thought to link GABAA receptors to the cytoskeleton. The multifunctional protein gephyrin represents a major component of the subsynaptic protein scaffold of inhibitory synapses and provides an interface for interaction with diverse other postsynaptic proteins including components of the microtubule and actin cytoskleton, as well as the GDP-GTP exchange factor collybistin, which is implicated in postsynaptic deposition of gephyrin. Analysis of gamma;2 subunit-deficient mice and neurons revealed that the gamma;2 subunit is essential for postsynaptic clustering of GABA A receptors and gephyrin, but largely dispensable for expression of functional GABA-gated chloride channels at the cell surface (Essrich et al., 1998). Interaction of the gamma;2 subunit with diverse putative trafficking proteins of GABAA receptors, such as GABARAP and GODZ, further suggests that this subunit acts as an important determinant of postsynaptic receptor concentration. Nevertheless, the mechanism by which the gamma;2 subunit contributes to accumulation of GABAA receptors at synapses is poorly understood. The main objective of this doctoral thesis was to determine the subunit domain(s) of the gamma;2 subunit that are essential (i) for proper trafficking and localization of postsynaptic GABAA receptors; (ii) for recruitment of gephyrin to postsynaptic GABAA receptors; and (iii) for normal inhibitory synaptic function of postsynaptic GABAA receptors in gamma;2 subunit-deficient neurons. Surprisingly, the fourth transmembrane domain (TM4) of the gamma;2 subunit was found to be sufficient for postsynaptic localization of GABAA receptors. However, the cytoplasmic loop domain is required in addition to TM4 for recruitment of gephyrin to postsynaptic GABAA receptors and for restoration of inhibitory synaptic function. These experiments point to a novel mechanism in subcellular targeting of ligand-gated ion channels and clearly dissociate postsynaptic GABAA receptor targeting mechanisms from interaction with gephyrin. As part of a collaborative project with Dr. Harvey's group at UC London, I was involved in mapping the protein-protein interaction domains between gephyrin and the GTP exchange factor collybistin to further elucidate their roles in synaptic localization and anchoring of GABAA receptors at the postsynaptic membrane. These experiments revealed that proper localization of gephyrin requires both the plextrin homology domain of collybistin and the collybistin binding sequence in gephyrin. Additionally, a single point mutation in the SH3 domain of collybistin known to underlie an atypical form of hyperekplexia in humans was shown to result in mislocalization of gephyrin and postsynaptic GABAA receptors in transfected neurons. These experiments for the first time showed an essential function of collybistin in formation of GABAergic inhibitory synapses. Finally, preliminary results addressing the role of palmitoylation of the gamma;2 subunit of GABAA receptors with respect to postsynaptic localization of GABAA receptors suggest that proper localization of GABAA receptors can occur independently of palmitoylation. Rather than proper localization, palmitoylation is therefore implicated in regulating the stability of GABAA receptors in the plasma membrane.

Modulation of the concentration of postsynaptic GABA(A) receptors contributes to functional plasticity of inhibitory synapses. The gamma2 subunit of GABA(A) receptor is specifically required for clustering of these receptors, for recruitment of the submembrane scaffold protein gephyrin to postsynaptic sites, and for postsynaptic function of GABAergic inhibitory synapses. To elucidate this mechanism, we here have mapped the gamma2 subunit domains required for restoration of postsynaptic clustering and function of GABA(A) receptors in gamma2 subunit mutant neurons. Transfection of gamma2-/- neurons with the gamma2 subunit but not the alpha2 subunit rescues postsynaptic clustering of GABA(A) receptors, results in recruitment of gephyrin to postsynaptic sites, and restores the amplitude and frequency of miniature inhibitory postsynaptic currents to wild-type levels. Analogous analyses of chimeric gamma2/alpha2 subunit constructs indicate, unexpectedly, that the fourth transmembrane domain of the gamma2 subunit is required and sufficient for postsynaptic clustering of GABA(A) receptors, whereas cytoplasmic gamma2 subunit domains are dispensable. In contrast, both the major cytoplasmic loop and the fourth transmembrane domain of the gamma2 subunit contribute to efficient recruitment of gephyrin to postsynaptic receptor clusters and are essential for restoration of miniature IPSCs. Our study points to a novel mechanism involved in targeting of GABA(A) receptors and gephyrin to inhibitory synapses

The neurotransmitter GABA activates heteropentameric GABA(A) receptors, which are composed mostly of alpha, beta, and gamma2 subunits. Regulated membrane trafficking and subcellular targeting of GABA(A) receptors is important for determining the efficacy of GABAergic inhibitory function. Of special interest is the gamma2 subunit, which is mostly dispensable for assembly and membrane insertion of functional receptors but essential for accumulation of GABA(A) receptors at synapses. In a search for novel receptor trafficking proteins, we have used the SOS-recruitment system and isolated a Golgi-specific DHHC zinc finger protein (GODZ) as a novel gamma2 subunit-interacting protein. GODZ is a member of the superfamily of DHHC cysteine-rich domain (DHHC-CRD) polytopic membrane proteins shown recently in yeast to represent palmitoyltransferases. GODZ mRNA is found in many tissues; however, in brain the protein is detected in neurons only and highly concentrated and asymmetrically distributed in the Golgi complex. GODZ interacts with a cysteine-rich 14-amino acid domain conserved specifically in the large cytoplasmic loop of gamma1-3 subunits but not in other GABA(A) receptor subunits. Coexpression of GODZ and GABA(A) receptors in heterologous cells results in palmitoylation of the gamma2 subunit in a cytoplasmic loop domain-dependent manner. Neuronal GABA(A) receptors are similarly palmitoylated. Thus, GODZ-mediated palmitoylation represents a novel posttranslational modification that is selective for gamma subunit-containing GABA(A) receptor subtypes, a mechanism that is likely to be important for regulated trafficking of these receptors in the secretory pathway

Glycine receptors (GlyRs) and specific subtypes of GABA(A) receptors are clustered at synapses by the multidomain protein gephyrin, which in turn is translocated to the cell membrane by the GDP-GTP exchange factor collybistin. We report the characterization of several new variants of collybistin, which are created by alternative splicing of exons encoding an N-terminal src homology 3 (SH3) domain and three alternate C termini (CB1, CB2, and CB3). The presence of the SH3 domain negatively regulates the ability of collybistin to translocate gephyrin to submembrane microaggregates in transfected mammalian cells. Because the majority of native collybistin isoforms appear to harbor the SH3 domain, this suggests that collybistin activity may be regulated by protein-protein interactions at the SH3 domain. We localized the binding sites for collybistin and the GlyR beta subunit to the C-terminal MoeA homology domain of gephyrin and show that multimerization of this domain is required for collybistin-gephyrin and GlyR-gephyrin interactions. We also demonstrate that gephyrin clustering in recombinant systems and cultured neurons requires both collybistin-gephyrin interactions and an intact collybistin pleckstrin homology domain. The vital importance of collybistin for inhibitory synaptogenesis is underlined by the discovery of a mutation (G55A) in exon 2 of the human collybistin gene (ARHGEF9) in a patient with clinical symptoms of both hyperekplexia and epilepsy. The clinical manifestation of this collybistin missense mutation may result, at least in part, from mislocalization of gephyrin and a major GABA(A) receptor subtype