Katharine R. Smith

Regulation of synaptic inhibition by phospho-dependent binding of the AP2 complex to a YECL motif in the GABAA receptor γ2 subunit

The regulation of the number of γ2-subunit-containing GABAA receptors (GABAARs) present at synapses is critical for correct synaptic inhibition and animal behavior. This regulation occurs, in part, by the controlled removal of receptors from the membrane in clathrin-coated vesicles, but it remains unclear how clathrin recruitment to surface γ2-subunit-containing GABAARs is regulated. Here, we identify a γ2-subunit-specific Yxxφ-type-binding motif for the clathrin adaptor protein, AP2, which is located within a site for γ2-subunit tyrosine phosphorylation. Blocking GABAAR-AP2 interactions via this motif increases synaptic responses within minutes. Crystallographic and biochemical studies reveal that phosphorylation of the Yxxφ motif inhibits AP2 binding, leading to increased surface receptor number. In addition, the crystal structure provides an explanation for the high affinity of this motif for AP2 and suggests that γ2-subunit-containing heteromeric GABAARs may be internalized as dimers or multimers. These data define a mechanism for tyrosine kinase regulation of GABAAR surface levels and synaptic inhibition.

NMDA receptors regulate GABAA receptor lateral mobility and clustering at inhibitory synapses through serine 327 on the γ2 subunit.

Modification of the number of GABAA receptors (GABAARs) clustered at inhibitory synapses can regulate inhibitory synapse strength with important implications for information processing and nervous system plasticity and pathology. Currently, however, the mechanisms that regulate the number of GABAARs at synapses remain poorly understood. By imaging superecliptic pHluorin tagged GABAAR subunits we show that synaptic GABAAR clusters are normally stable, but that increased neuronal activity upon glutamate receptor (GluR) activation results in their rapid and reversible dispersal. This dispersal correlates with increases in the mobility of single GABAARs within the clusters as determined using single-particle tracking of GABAARs labeled with quantum dots. GluR-dependent dispersal of GABAAR clusters requires Ca2+ influx via NMDA receptors (NMDARs) and activation of the phosphatase calcineurin. Moreover, the dispersal of GABAAR clusters and increased mobility of individual GABAARs are dependent on serine 327 within the intracellular loop of the GABAAR γ2 subunit. Thus, NMDAR signaling, via calcineurin and a key GABAAR phosphorylation site, controls the stability of synaptic GABAARs, with important implications for activity-dependent control of synaptic inhibition and neuronal plasticity.

Psychiatric risk factor ANK3/Ankyrin-G nanodomains regulate the structure and function of glutamatergic synapses

Recent evidence implicates glutamatergic synapses as key pathogenic sites in psychiatric disorders. Common and rare variants in the ANK3 gene, encoding ankyrin-G, have been associated with bipolar disorder, schizophrenia, and autism. Here we demonstrate that ankyrin-G is integral to AMPAR-mediated synaptic transmission and maintenance of spine morphology. Using superresolution microscopy we find that ankyrin-G forms distinct nanodomain structures within the spine head and neck. At these sites, it modulates mushroom spine structure and function, probably as a perisynaptic scaffold and barrier within the spine neck. Neuronal activity promotes ankyrin-G accumulation in distinct spine subdomains, where it differentially regulates NMDA receptor-dependent plasticity. These data implicate subsynaptic nanodomains containing a major psychiatric risk molecule, ankyrin-G, as having location-specific functions and open directions for basic and translational investigation of psychiatric risk molecules.

An Autism-Associated Variant of Epac2 Reveals a Role for Ras/Epac2 Signaling in Controlling Basal Dendrite Maintenance in Mice

Epac2 disruption impairs basal (but not apical) dendrite complexity in cortical neurons, and an autism-associated mutation in Epac2 implicates a Ras/Epac2 signaling pathway in the active maintenance of basal dendritic arbors.

Shank3 Deficiency Induces NMDA Receptor Hypofunction via an Actin-Dependent Mechanism

Shank3, which encodes a scaffolding protein at glutamatergic synapses, is a genetic risk factor for autism. In this study, we examined the impact of Shank3 deficiency on the NMDA-type glutamate receptor, a key player in cognition and mental illnesses. We found that knockdown of Shank3 with a small interfering RNA (siRNA) caused a significant reduction of NMDAR-mediated ionic or synaptic current, as well as the surface expression of NR1 subunits, in rat cortical cultures. The effect of Shank3 siRNA on NMDAR currents was blocked by an actin stabilizer, and was occluded by an actin destabilizer, suggesting the involvement of actin cytoskeleton. Since actin dynamics is regulated by the GTPase Rac1 and downstream effector p21-activated kinase (PAK), we further examined Shank3 regulation of NMDARs when Rac1 or PAK was manipulated. We found that the reducing effect of Shank3 siRNA on NMDAR currents was mimicked and occluded by specific inhibitors for Rac1 or PAK, and was blocked by constitutively active Rac1 or PAK. Immunocytochemical data showed a strong reduction of F-actin clusters after Shank3 knockdown, which was occluded by a PAK inhibitor. Inhibiting cofilin, the primary downstream target of PAK and a major actin depolymerizing factor, prevented Shank3 siRNA from reducing NMDAR currents and F-actin clusters. Together, these results suggest that Shank3 deficiency induces NMDAR hypofunction by interfering with the Rac1/PAK/cofilin/actin signaling, leading to the loss of NMDAR membrane delivery or stability. It provides a potential mechanism for the role of Shank3 in cognitive deficit in autism.

Stabilization of GABAA Receptors at Endocytic Zones Is Mediated by an AP2 Binding Motif within the GABAA Receptor β3 Subunit

The strength of synaptic inhibition can be controlled by the stability and endocytosis of surface and synaptic GABAA receptors (GABAARs), but the surface receptor dynamics that underpin GABAAR recruitment to dendritic endocytic zones (EZs) have not been investigated. Stabilization of GABAARs at EZs is likely to be regulated by receptor interactions with the clathrin-adaptor AP2, but the molecular determinants of these associations remain poorly understood. Moreover, although surface GABAAR downmodulation plays a key role in pathological disinhibition in conditions such as ischemia and epilepsy, whether this occurs in an AP2-dependent manner also remains unclear. Here we report the characterization of a novel motif containing three arginine residues (405RRR407) within the GABAAR β3-subunit intracellular domain (ICD), responsible for the interaction with AP2 and GABAAR internalization. When this motif is disrupted, binding to AP2 is abolished in vitro and in rat brain. Using single-particle tracking, we reveal that surface β3-subunit-containing GABAARs exhibit highly confined behavior at EZs, which is dependent on AP2 interactions via this motif. Reduced stabilization of mutant GABAARs at EZs correlates with their reduced endocytosis and increased steady-state levels at synapses. By imaging wild-type or mutant super-ecliptic pHluorin-tagged GABAARs in neurons, we also show that, under conditions of oxygen–glucose deprivation to mimic cerebral ischemia, GABAARs are depleted from synapses in dendrites, depending on the 405RRR407 motif. Thus, AP2 binding to an RRR motif in the GABAAR β3-subunit ICD regulates GABAAR residency time at EZs, steady-state synaptic receptor levels, and pathological loss of GABAARs from synapses during simulated ischemia.

GIT1 and βPIX Are Essential for GABAA Receptor Synaptic Stability and Inhibitory Neurotransmission

Summary Effective inhibitory synaptic transmission requires efficient stabilization of GABAA receptors (GABAARs) at synapses, which is essential for maintaining the correct excitatory-inhibitory balance in the brain. However, the signaling mechanisms that locally regulate synaptic GABAAR membrane dynamics remain poorly understood. Using a combination of molecular, imaging, and electrophysiological approaches, we delineate a GIT1/βPIX/Rac1/PAK signaling pathway that modulates F-actin and is important for maintaining surface GABAAR levels, inhibitory synapse integrity, and synapse strength. We show that GIT1 and βPIX are required for synaptic GABAAR surface stability through the activity of the GTPase Rac1 and downstream effector PAK. Manipulating this pathway using RNAi, dominant-negative and pharmacological approaches leads to a disruption of GABAAR clustering and decrease in the strength of synaptic inhibition. Thus, the GIT1/βPIX/Rac1/PAK pathway plays a crucial role in regulating GABAAR synaptic stability and hence inhibitory synaptic transmission with important implications for inhibitory plasticity and information processing in the brain.

The cell biology of synaptic inhibition in health and disease

Fast synaptic inhibition is largely mediated by GABA(A) receptors (GABA(A)Rs), ligand-gated chloride channels that play an essential role in the control of cell and network activity in the brain. Recent work has demonstrated that the delivery, number and stability of GABA(A)Rs at inhibitory synapses play a key role in the dynamic regulation of inhibitory synaptic efficacy and plasticity. The regulatory pathways essential for the fine-tuning of synaptic inhibition have also emerged as key sites of vulnerability during pathological changes in cell excitability in disease states.

Regulation of inhibitory synaptic transmission by a conserved atypical interaction of GABAA receptor β- and γ-subunits with the clathrin AP2 adaptor

The number of surface and synaptic GABA(A) receptors is an important determinant of inhibitory synapse strength. Surface receptor number is in part controlled by removal of receptors from the membrane by interaction with the clathrin adaptor AP2. Here we demonstrate that there are two binding sites for AP2 in the gamma2-subunit: a Yxxvarphi type motif specific to gamma2-subunits and a basic patch AP2 binding motif, that is also found in GABA(A) receptor beta-subunits. Blocking GABA(A) receptor-AP2 interactions using a peptide that inhibits AP2 binding to GABA(A) receptors via the conserved basic patch mechanism increases synaptic responses within minutes, whereas simultaneously blocking both binding mechanisms has an additive effect. These data suggest that multiple AP2 internalization signals control the levels of surface and synaptic GABA(A) receptors to regulate synaptic inhibition.

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.