Phospho-dependent binding of the clathrin AP2 adaptor complex to GABA _A receptors regulates the efficacy of inhibitory synaptic transmission

Abstract

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...

Molecular determinants for the interaction between AMPA receptors and the clathrin adaptor complex AP-2

“…In agreement with the observation that GluR2 exerts dominant effects over GluR1 with regard to AMPA receptor internalization (15), we have found that AP-2 appears to interact much more efficiently with GluR2 or GluR3 than with GluR1 CTs. Moreover, we have mapped the GluR2 binding activity to a site within 2 that is also responsible for its binding to inhibitory postsynaptic GABA A receptors (17) and to the presynaptic vesicle protein synaptotagmin (9,18), suggesting a common mechanism for recognition of pre-and postsynaptic membrane cargo proteins by AP-2. This view is also supported by our observation that increasing concentrations of inositolpolyphosphates inhibit the association of GluR2 with AP-2, similar to what has been reported before for the synaptotagmin 1-AP-2 complex (10).…”

“…The observation that various pre-and postsynaptic membrane receptors including AMPA and GABA A receptors (17), as well as synaptotagmin family members (9) use a common mechanism for interacting with a cognate recognition site within subdomain B of 2-adaptin (17,18) poses the question of how exactly this is accomplished and why a distinct atypical internalization signal is used for this type of cargo proteins. One possibility could be that atypical endocytosis signals within synaptic cargo membrane proteins could contribute to their sorting into specialized endosomes.…”

Dopamine D3Receptors Regulate GABAAReceptor Function through a Phospho-Dependent Endocytosis Mechanism in Nucleus Accumbens

“…The peptide pep␤3 was synthesized corresponding to residues 401-412 (KTHLRRRSSQLK) of the rat GABA A R ␤3 subunit. An identical peptide (pep␤-phos) chemically phosphorylated at S408/S409 was synthesized as described previously (Kittler et al, 2005). The peptide pep␤3-[S3 A] was synthesized based on the same amino acid sequence as pep␤3, except that S408/S409 were both changed to Ala (KTHLR-RRAAQLK).…”

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

“…For OGD treatment ACSF was replaced with OGD buffer (126 mM NaCl, 24 mM NaHCO 3, 1 mM NaH2PO4, 2.5 mM KCl, 1 mM MgCl2, 2 mM CaCl 2, 7 mM sucrose, and gassed with 95% N2/5% CO2) (42 Electrophysiology. Whole-cell recordings of mIPSCs from cortical slices, or GFP-2FYVE transfected cultured cortical cells, were performed using standard voltage clamp techniques in the presence of CNQX (20 M) and APV (40 M) to block AMPA and NMDA receptors, respectively (43,44). Membrane currents from HEK-293 cells were measured, as outlined previously at 32°C (10,43,45).…”

“…Whole-cell recordings of mIPSCs from cortical slices, or GFP-2FYVE transfected cultured cortical cells, were performed using standard voltage clamp techniques in the presence of CNQX (20 M) and APV (40 M) to block AMPA and NMDA receptors, respectively (43,44). Membrane currents from HEK-293 cells were measured, as outlined previously at 32°C (10,43,45). Data were collected 5 min after obtaining a stable recording conformation.…”