RESUMO
The regulation of the number of gamma2-subunit-containing GABA(A) receptors (GABA(A)Rs) 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 gamma2-subunit-containing GABA(A)Rs is regulated. Here, we identify a gamma2-subunit-specific Yxxvarphi-type-binding motif for the clathrin adaptor protein, AP2, which is located within a site for gamma2-subunit tyrosine phosphorylation. Blocking GABA(A)R-AP2 interactions via this motif increases synaptic responses within minutes. Crystallographic and biochemical studies reveal that phosphorylation of the Yxxvarphi 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 gamma2-subunit-containing heteromeric GABA(A)Rs may be internalized as dimers or multimers. These data define a mechanism for tyrosine kinase regulation of GABA(A)R surface levels and synaptic inhibition.
Assuntos
Complexo 2 de Proteínas Adaptadoras/metabolismo , Subunidades Proteicas/metabolismo , Receptores de GABA-A/metabolismo , Complexo 2 de Proteínas Adaptadoras/química , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Sítios de Ligação , Cristalografia por Raios X , Endocitose , Fosforilação , Ligação Proteica/fisiologia , Conformação Proteica , Subunidades Proteicas/fisiologia , Ratos , Receptores de GABA-A/química , Receptores de GABA-A/fisiologia , SinapsesRESUMO
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.
Assuntos
Complexo 2 de Proteínas Adaptadoras/fisiologia , Potenciais Pós-Sinápticos Inibidores/fisiologia , Inibição Neural/fisiologia , Neurônios/fisiologia , Receptores de GABA-A/fisiologia , Complexo 2 de Proteínas Adaptadoras/química , Animais , Sítios de Ligação/efeitos dos fármacos , Corpo Estriado/citologia , Técnicas In Vitro , Potenciais Pós-Sinápticos Inibidores/efeitos dos fármacos , Inibição Neural/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Técnicas de Patch-Clamp , Domínios e Motivos de Interação entre Proteínas/fisiologia , Estrutura Terciária de Proteína/fisiologia , Subunidades Proteicas/química , Subunidades Proteicas/fisiologia , Ratos , Receptores de GABA-A/química , Alinhamento de Sequência , Sinaptotagmina I/farmacologia , Fatores de TempoRESUMO
Fast synaptic inhibition in the brain is largely mediated by ionotropic GABA receptors, which can be subdivided into GABAA and GABAC receptors based on pharmacological and molecular criteria. GABAA receptors are important therapeutic targets for a range of sedative, anxiolytic, and hypnotic agents and are implicated in several diseases including epilepsy, anxiety, depression, and substance abuse. In addition, modulating the efficacy of GABAergic neurotransmission may play a key role in neuronal plasticity. Recent studies have begun to reveal that the accumulation of ionotropic GABAA receptors at synapses is a highly regulated process that is facilitated by receptor-associated proteins and other cell-signaling molecules. This review focuses on recent experimental evidence detailing the mechanisms that control the assembly and transport of functional ionotropic GABAA receptors to cell surface sites, in addition to their stability at synaptic sites. These regulatory processes will be discussed within the context of the dynamic modulation of synaptic inhibition in the central nervous system (CNS).
Assuntos
Inibição Neural/fisiologia , Processamento de Proteína Pós-Traducional/fisiologia , Receptores de GABA-A/química , Receptores de GABA-A/metabolismo , Transmissão Sináptica/fisiologia , Animais , Humanos , Transporte Proteico/fisiologiaRESUMO
The efficacy of synaptic inhibition depends on the number of gamma-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 GABA(A)R endocytosis and, hence, surface receptor number. Here, we identify a previously uncharacterized atypical AP2 binding motif conserved within the intracellular domains of all GABA(A)R beta subunit isoforms. This AP2 binding motif (KTHLRRRSSQLK in the beta3 subunit) incorporates the major sites of serine phosphorylation within receptor beta subunits, and phosphorylation within this site inhibits AP2 binding. Furthermore, by using surface plasmon resonance, we establish that a peptide (pepbeta3) corresponding to the AP2 binding motif in the GABA(A)R beta3 subunit binds to AP2 with high affinity only when dephosphorylated. Moreover, the pepbeta3 peptide, but not its phosphorylated equivalent (pepbeta3-phos), enhanced the amplitude of miniature inhibitory synaptic current and whole cell GABA(A)R current. These effects of pepbeta3 on GABA(A)R current were occluded by inhibitors of dynamin-dependent endocytosis supporting an action of pepbeta3 on GABA(A)R endocytosis. Therefore phospho-dependent regulation of AP2 binding to GABA(A)Rs provides a mechanism to specify receptor cell surface number and the efficacy of inhibitory synaptic transmission.