The efficacy of fast synaptic inhibition is critically dependent on the accumulation of GABA A receptors at inhibitory synapses, a process that remains poorly understood. Here, we examined the dynamics of cell surface GABA A receptors using receptor subunits modified with N-terminal extracellular ecliptic pHluorin reporters. In hippocampal neurons, GABA A receptors incorporating pHluorin-tagged subunits were found to be clustered at synaptic sites and also expressed as diffuse extrasynaptic staining. By combining FRAP (fluorescence recovery after photobleaching) measurements with live imaging of FM4-64-labeled active presynaptic terminals, it was evident that clustered synaptic receptors exhibit significantly lower rates of mobility at the cell surface compared with their extrasynaptic counterparts. To examine the basis of this confinement, we used RNAi to inhibit the expression of gephyrin, a protein shown to regulate the accumulation of GABA A receptors at synaptic sites. However, whether gephyrin acts to control the actual formation of receptor clusters, their stability, or is simply a global regulator of receptor cell surface number remains unknown. Inhibiting gephyrin expression did not modify the total number of GABA A receptors expressed on the neuronal cell surface but significantly decreased the number of receptor clusters. Live imaging revealed that clusters that formed in the absence of gephyrin were significantly more mobile compared with those in control neurons. Together, our results demonstrate that synaptic GABA A receptors have lower levels of lateral mobility compared with their extrasynaptic counterparts, and suggest a specific role for gephyrin in reducing the diffusion of GABA A receptors, facilitating their accumulation at inhibitory synapses.
The efficacy of synaptic inhibition depends on the number of ␥-aminobutyric acid type A receptors (GABA A Rs) expressed on the cell surface of neurons. The clathrin adaptor protein 2 (AP2) complex is a critical regulator of GABAAR endocytosis and, hence, surface receptor number. Here, we identify a previously uncharacterized atypical AP2 binding motif conserved within the intracellular domains of all GABAAR  subunit isoforms. This AP2 binding motif (KTHLRRRSSQLK in the 3 subunit) incorporates the major sites of serine phosphorylation within receptor  subunits, and phosphorylation within this site inhibits AP2 binding. Furthermore, by using surface plasmon resonance, we establish that a peptide (pep3) corresponding to the AP2 binding motif in the GABAAR 3 subunit binds to AP2 with high affinity only when dephosphorylated. Moreover, the pep3 peptide, but not its phosphorylated equivalent (pep3-phos), enhanced the amplitude of miniature inhibitory synaptic current and whole cell GABAAR current. These effects of pep3 on GABAAR current were occluded by inhibitors of dynamin-dependent endocytosis supporting an action of pep3 on GABAAR endocytosis. Therefore phosphodependent regulation of AP2 binding to GABAARs provides a mechanism to specify receptor cell surface number and the efficacy of inhibitory synaptic transmission. endocytosis ͉ phosphorylation G ABA A receptors (GABA A Rs) are the major sites of fast synaptic inhibition in the brain (1). These pentameric ligandgated ion channels can be constructed from seven subunit classes: ␣1-6,  1-3, ␥ 1-3, ␦, , , and (2), with the majority of benzodiazepine-sensitive receptor subtypes being assembled from ␣, , and ␥2 subunits (1, 2). A primary determinant for the efficacy of synaptic inhibition and, hence, neuronal excitation is the number of functional GABA A Rs expressed on the surface of neurons (3-10). Therefore, there has been considerable interest in understanding the cellular mechanism that neurons use to regulate GABA A R cell surface stability and activity. Collectively these studies have revealed that neuronal GABA A Rs undergo significant rates of constitutive endocytosis (3,8,(11)(12)(13)(14)(15), a process that has been established to regulate synaptic inhibition (8). GABA A Rs enter the endocytic pathway by a clathrin-mediated dynamindependent mechanism (8,(11)(12)(13)(14), a process that is facilitated by the clathrin adaptor protein 2 (AP2) complex, which is intimately associated with these receptors in neurons (8, 13). Internalized GABA A Rs are then subjected to either rapid recycling or targeted for lysozomal degradation, an endoctytic sorting decision that is regulated by the Huntingtin associated protein-1 (15). Therefore, changes in the rates of GABA A R endocytosis and͞or endocytic sorting represent potentially powerful mechanisms to regulate GABA A R cell surface number and inhibitory synaptic transmission (8,15).A potential mechanism to regulate target protein endocytosis is modulating interaction with the AP2 adaptor protein complex (1...
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