RESUMO
AMPA (alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid) receptor trafficking is a fundamental mechanism for regulating synaptic strength, and hence may underlie cellular processes involved in learning and memory. PICK1 (protein that interacts with protein C-kinase) has recently emerged as a key regulator of AMPAR (AMPA receptor) traffic, and the precise molecular mechanisms of PICK1's action are just beginning to be unravelled. In this review, I summarize recent findings that describe some important molecular characteristics of PICK1 with respect to AMPAR cell biology.
Assuntos
Proteínas de Transporte/fisiologia , Proteínas Nucleares/fisiologia , Receptores de AMPA/fisiologia , Regulação Alostérica , Animais , Cálcio/fisiologia , Sinalização do Cálcio , Membrana Celular/fisiologia , Membrana Celular/ultraestrutura , Homeostase , Potenciação de Longa Duração , Depressão Sináptica de Longo Prazo , Modelos Moleculares , Conformação Proteica , Transporte Proteico , Receptores de AMPA/metabolismoRESUMO
Ionotropic gamma-aminobutyric acid (GABA(A) and GABA(C)) receptors mediate fast synaptic inhibition in the central nervous system. GABA(C) receptors are expressed predominantly in the retina on bipolar cell axon terminals, and are thought to mediate feedback inhibition from GABAergic amacrine cells. Utilizing the yeast two-hybrid system, we previously identified MAP1B as a binding partner of the GABA(C) receptor rho1 subunit. Here we describe the isolation of an additional rho1 interacting protein: a novel C-terminal variant of the glycine transporter GLYT-1. We show that GLYT-1 exists as four alternatively spliced mRNAs which encode proteins expressing one of two possible intracellullar N- and C-terminal domains. Variants containing the novel C terminus efficiently transport glycine when expressed in COS cells, but with unusual kinetics. We have confirmed the interaction between the novel C terminus and rho1 subunit and demonstrated binding in heterologous cells. This interaction may be crucial for the integration of GABAergic and glycinergic neurotransmission in the retina.
Assuntos
Processamento Alternativo , Sistemas de Transporte de Aminoácidos Neutros , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Glicina/metabolismo , Receptores de GABA/metabolismo , Retina/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Células COS , Proteínas de Transporte/química , Bovinos , Clonagem Molecular , Primers do DNA , Biblioteca Gênica , Variação Genética , Proteínas da Membrana Plasmática de Transporte de Glicina , Dados de Sequência Molecular , RNA Mensageiro/genética , Receptores de GABA/química , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae , Alinhamento de Sequência , Sódio/metabolismo , TransfecçãoRESUMO
GABA(C) receptors contain rho subunits and mediate feedback inhibition from retinal amacrine cells to bipolar cells. We previously identified the cytoskeletal protein MAP1B as a rho1 subunit anchoring protein. Here, we analyze the structural basis and functional significance of the MAP1B-rho1 interaction. Twelve amino acids at the C terminus of the large intracellular loop of rho1 (and also rho2) are sufficient for interaction with MAP1B. Disruption of the MAP1B-rho interaction in bipolar cells in retinal slices decreased the EC(50) of their GABA(C) receptors, doubling the receptors' current at low GABA concentrations without affecting their maximum current at high concentrations. Thus, anchoring to the cytoskeleton lowers the sensitivity of GABA(C) receptors and provides a likely site for functional modulation of GABA(C) receptor-mediated inhibition.
Assuntos
Sistemas de Transporte de Aminoácidos Neutros , Proteínas Associadas aos Microtúbulos/metabolismo , Receptores de GABA-B , Receptores de GABA/metabolismo , Animais , Sítios de Ligação/genética , Ligação Competitiva/efeitos dos fármacos , Ligação Competitiva/genética , Western Blotting , Células COS , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Células Cultivadas , Relação Dose-Resposta a Droga , Antagonistas GABAérgicos/farmacologia , Glutationa Transferase/genética , Glicinérgicos/farmacologia , Proteínas da Membrana Plasmática de Transporte de Glicina , Técnicas In Vitro , Proteínas Associadas aos Microtúbulos/genética , Mutagênese Sítio-Dirigida , Técnicas de Patch-Clamp , Peptídeos/genética , Peptídeos/farmacologia , Ácidos Fosfínicos/farmacologia , Estrutura Terciária de Proteína/genética , Piridinas/farmacologia , Receptores de GABA/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Retina/efeitos dos fármacos , Retina/metabolismo , Transfecção , Ácido gama-Aminobutírico/metabolismo , Ácido gama-Aminobutírico/farmacocinéticaRESUMO
The ionotropic type-A and type-C receptors for the neurotransmitter gamma-aminobutyric acid (GABA(A) and GABA(C) receptors) are the principal sites of fast synaptic inhibition in the central nervous system, but it is not known how these receptors are localized at GABA-dependent synapses. GABA(C) receptors, which are composed of rho-subunits, are expressed almost exclusively in the retina of adult vertebrates, where they are enriched on bipolar cell axon terminals. Here we show that the microtubule-associated protein 1B (MAP-1B) specifically interacts with the GABA(C) rho1 subunit but not with GABA(A) receptor subunits. Furthermore, GABA(C) receptors and MAP-1B co-localize at postsynaptic sites on bipolar cell axon terminals. Co-expression of MAP-1B and the rho1 subunit in COS cells results in a dramatic redistribution of the rho1 subunit. Our observations suggest a novel mechanism for localizing ionotropic GABA receptors to synaptic sites. This mechanism, which is specific for GABA(C) but not GABA(A) receptors, may allow these receptor subtypes, which have distinct physiological and pharmacological properties, to be differentially localized at inhibitory synapses.
