Proteomic Characterization of Inhibitory Synapses Using a Novel pHluorin-tagged γ-Aminobutyric Acid Receptor, Type A (GABAA), α2 Subunit Knock-in Mouse.

The accumulation of γ-aminobutyric acid receptors (GABAARs) at the appropriate postsynaptic sites is critical for determining the efficacy of fast inhibitory neurotransmission. Although we know that the majority of synaptic GABAAR subtypes are assembled from α1-3, ß, and γ2 subunits, our understanding of how neurons facilitate their targeting to and stabilization at inhibitory synapses is rudimentary. To address these issues, we have created knock-in mice in which the pH-sensitive green fluorescent protein (GFP) and the Myc epitope were introduced to the extracellular domain of the mature receptor α2 subunit (pHα2). Using immunoaffinity purification and mass spectroscopy, we identified a stable complex of 174 proteins that were associated with pHα2, including other GABAAR subunits, and previously identified receptor-associated proteins such as gephyrin and collybistin. 149 of these proteins were novel GABAAR binding partners and included G-protein-coupled receptors and ion channel subunits, proteins that regulate trafficking and degradation, regulators of protein phosphorylation, GTPases, and a number of proteins that regulate their activity. Notably, members of the postsynaptic density family of proteins that are critical components of excitatory synapses were not associated with GABAARs. Crucially, we demonstrated for a subset of these novel proteins (including cullin1, ephexin, potassium channel tetramerization domain containing protein 12, mitofusin2, metabotropic glutamate receptor 5, p21-activated kinase 7, and Ras-related protein 5A) bind directly to the intracellular domains of GABAARs, validating our proteomic analysis. Thus, our experiments illustrate the complexity of the GABAAR proteome and enhance our understanding of the mechanisms neurons use to construct inhibitory synapses.

Benzodiazepines modulate GABAA receptors by regulating the preactivation step after GABA binding.

GABA(A) receptors (GABA(A)Rs) composed of αßγ subunits are allosterically modulated by the benzodiazepines (BDZs). Agonists at the BDZ binding site potentiate submaximal GABA responses by increasing the apparent affinity of GABA(A)Rs for GABA. Although BDZs were initially thought to affect the binding of GABA agonists, recent studies suggest an effect on receptor gating; however, the involvement of preactivation steps in the modulation by BDZs has not been considered. Consequently, we examined whether BDZ agonists could exert their modulatory effect by displacing the equilibrium between resting and preactivated states of recombinant α1ß2γ2 GABA(A)Rs expressed in Xenopus oocytes. For GABA and the partial agonists 4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol and piperidine-4-sulfonic acid, we examined BDZ modulation using a simple three-step model incorporating agonist binding, receptor preactivation, and channel opening. The model accounted for diazepam modulation simply by increasing the preactivation constant by approximately fourfold. To assess whether BDZs preferentially affected a specific GABA binding site, pentameric concatamers were used. This demonstrated that single GABA-binding site mutant receptors were equally sensitive to modulation by BDZs compared with wild-type counterparts. Overall, our results suggest that BDZs affect the preactivation step to cause a global conformational rearrangement of GABA(A)Rs, thereby modulating receptor function.

Ubiquitin-dependent lysosomal targeting of GABA(A) receptors regulates neuronal inhibition.

The strength of synaptic inhibition depends partly on the number of GABA(A) receptors (GABA(A)Rs) found at synaptic sites. The trafficking of GABA(A)Rs within the endocytic pathway is a key determinant of surface GABA(A)R number and is altered in neuropathologies, such as cerebral ischemia. However, the molecular mechanisms and signaling pathways that regulate this trafficking are poorly understood. Here, we report the subunit specific lysosomal targeting of synaptic GABA(A)Rs. We demonstrate that the targeting of synaptic GABA(A)Rs into the degradation pathway is facilitated by ubiquitination of a motif within the intracellular domain of the gamma2 subunit. Blockade of lysosomal activity or disruption of the trafficking of ubiquitinated cargo to lysosomes specifically increases the efficacy of synaptic inhibition without altering excitatory currents. Moreover, mutation of the ubiquitination site within the gamma2 subunit retards the lysosomal targeting of GABA(A)Rs and is sufficient to block the loss of synaptic GABA(A)Rs after anoxic insult. Together, our results establish a previously unknown mechanism for influencing inhibitory transmission under normal and pathological conditions.

Identification and characterisation of a Maf1/Macoco protein complex that interacts with GABAA receptors in neurons.

The majority of fast inhibitory synaptic transmission in the mammalian nervous system is mediated by GABA(A) receptors (GABA(A)Rs). Here we report a novel interaction between the protein Maf1 and GABA(A)R beta-subunit intracellular domains. We find Maf1 to be highly expressed in brain and enriched in the hippocampus and cortex. In heterologous cells and neurons we show Maf1 co-localises with GABA(A)Rs in intracellular compartments and at the cell surface. In neurons, Maf1 is found localised in the cytoplasm in dendrites, partially overlapping with GABA(A)Rs and inhibitory synapses and in addition is enriched in the neuronal nucleus. We also report that Maf1 interacts with a novel coiled-coil domain containing protein that we have called Macoco (for Maf1 interacting coiled-coil protein). Like Maf1, Macoco can also be found localised to inhibitory synapses and directly interacts with GABA(A)Rs. Expressing Macoco in neurons increases surface GABA(A)R levels. Our results suggest that Maf1 and Macoco are novel GABA(A)R interacting proteins important for regulating GABA(A)R surface expression and GABA(A)R signalling in the brain.

Alanine:glyoxylate aminotransferase peroxisome-to-mitochondrion mistargeting in human hereditary kidney stone disease.

The pyridoxal-phosphate (PLP)-dependent enzyme alanine:glyoxylate aminotransferase (AGT) is mistargeted from peroxisomes to mitochondria in patients with the hereditary kidney stone disease primary hyperoxaluria type 1 (PH1) due to the synergistic interaction between a common Pro(11)Leu polymorphism and a PH1-specific Gly(170)Arg mutation. The kinetic partitioning of newly synthesised AGT between peroxisomes and mitochondria is determined by the combined effects of (1) the generation of cryptic mitochondrial targeting information, and (2) the inhibition of AGT dimerization. The crystal structure of AGT has recently been solved, allowing the effects of the various polymorphisms and mutations to be rationalised in terms of AGT's three-dimensional conformation. Procedures that increase dimer stability and/or increase the rate of dimer formation have potential in the formulation of novel strategies to treat this otherwise intractable life-threatening disease.

Delivery of GABAARs to synapses is mediated by HAP1-KIF5 and disrupted by mutant huntingtin.

The density of GABA(A) receptors (GABA(A)Rs) at synapses regulates brain excitability, and altered inhibition may contribute to Huntington's disease, which is caused by a polyglutamine repeat in the protein huntingtin. However, the machinery that delivers GABA(A)Rs to synapses is unknown. We demonstrate that GABA(A)Rs are trafficked to synapses by the kinesin family motor protein 5 (KIF5). We identify the adaptor linking the receptors to KIF5 as the huntingtin-associated protein 1 (HAP1). Disrupting the HAP1-KIF5 complex decreases synaptic GABA(A)R number and reduces the amplitude of inhibitory postsynaptic currents. When huntingtin is mutated, as in Huntington's disease, GABA(A)R transport and inhibitory synaptic currents are reduced. Thus, HAP1-KIF5-dependent GABA(A)R trafficking is a fundamental mechanism controlling the strength of synaptic inhibition in the brain. Its disruption by mutant huntingtin may explain some of the defects in brain information processing occurring in Huntington's disease and provides a molecular target for therapeutic approaches.

Peroxisomal import of human alanine:glyoxylate aminotransferase requires ancillary targeting information remote from its C terminus.

Although human alanine:glyoxylate aminotransferase (AGT) is imported into peroxisomes by a Pex5p-dependent pathway, the properties of its C-terminal tripeptide (KKL) are unlike those of any other type 1 peroxisomal targeting sequence (PTS1). We have previously suggested that AGT might possess ancillary targeting information that enables its unusual PTS1 to work. In this study, we have attempted to locate this information and to determine whether or not it is a characteristic of all vertebrate AGTs. Using the two-hybrid system, we show that human AGT interacts with human Pex5p in mammalian cells, but not yeast cells. Using (immuno)fluorescence microscopic analysis of the distribution of various constructs expressed in COS cells, we show the following. 1) The putative ancillary peroxisomal targeting information (PTS1A) in human AGT is located entirely within the smaller C-terminal structural domain of 110 amino acids, with the sequence between Val-324 and Ile-345 being the most likely candidate region. 2) The PTS1A is present in all mammalian AGTs studied (human, rat, guinea pig, rabbit, and cat), but not amphibian AGT (Xenopus). 3) The PTS1A is necessary for peroxisomal import of human, rabbit, and cat AGTs, but not rat and guinea pig AGTs. We speculate that the internal PTS1A of human AGT works in concert with the C-terminal PTS1 by interacting with Pex5p indirectly with the aid of a yet-to-be-identified mammal-specific adaptor molecule. This interaction might reshape the tetratricopeptide repeat domain allosterically, enabling it to accept KKL as a functional PTS1.

Correction of an enzyme trafficking defect in hereditary kidney stone disease in vitro.

In normal human hepatocytes, the intermediary-metabolic enzyme alanine:glyoxylate aminotransferase (AGT) is located within the peroxisomes. However, in approx. one-third of patients suffering from the hereditary kidney stone disease primary hyperoxaluria type 1, AGT is mistargeted to the mitochondria. AGT mistargeting results from the synergistic interaction between a common P11L (Pro11-->Leu) polymorphism and a disease-specific G170R mutation. The polymorphism generates a functionally weak mitochondrial targeting sequence, the efficiency of which is increased by the mutation. The two substitutions together, but not in isolation, inhibit AGT dimerization, highlighting the different structural requirements of the peroxisomal and mitochondrial protein-import machineries. In the present study, we show that treatments known to increase the stability of proteins non-specifically (i.e. lowering the temperature from 37 to 30 degrees C or by the addition of glycerol) completely normalize the intracellular targeting of mutant AGT expressed in transfected COS cells. On the other hand, treatments known to decrease protein stability (e.g. increasing the temperature from 37 to 42 degrees C) exacerbate the targeting defect. Neither of the treatments affects the relative efficiencies of the peroxisomal and mitochondrial protein-import pathways intrinsically. Results are discussed in the light of the known structural requirements of the two protein trafficking pathways and the formulation of possible treatment strategies for primary hyperoxaluria type 1.