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 (pep␤3) corresponding to the AP2 binding motif in the GABAAR ␤3 subunit binds to AP2 with high affinity only when dephosphorylated. Moreover, the pep␤3 peptide, but not its phosphorylated equivalent (pep␤3-phos), enhanced the amplitude of miniature inhibitory synaptic current and whole cell GABAAR current. These effects of pep␤3 on GABAAR current were occluded by inhibitors of dynamin-dependent endocytosis supporting an action of pep␤3 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...