RESUMEN
Newborn neurons in the dentate gyrus of the adult hippocampus rely upon cAMP response element binding protein (CREB) signaling for their differentiation into mature granule cells and their integration into the dentate network. Among its many targets, the transcription factor CREB activates expression of a gene locus that produces two microRNAs, miR-132 and miR-212. In cultured cortical and hippocampal neurons, miR-132 functions downstream from CREB to mediate activity-dependent dendritic growth and spine formation in response to a variety of signaling pathways. To investigate whether miR-132 and/or miR-212 contribute to the maturation of dendrites in newborn neurons in the adult hippocampus, we inserted LoxP sites surrounding the miR-212/132 locus and specifically targeted its deletion by stereotactically injecting a retrovirus expressing Cre recombinase. Deletion of the miR-212/132 locus caused a dramatic decrease in dendrite length, arborization, and spine density. The miR-212/132 locus may express up to four distinct microRNAs, miR-132 and -212 and their reverse strands miR-132* and -212*. Using ratiometric microRNA sensors, we determined that miR-132 is the predominantly active product in hippocampal neurons. We conclude that miR-132 is required for normal dendrite maturation in newborn neurons in the adult hippocampus and suggest that this microRNA also may participate in other examples of CREB-mediated signaling.
Asunto(s)
Dendritas/genética , Regulación de la Expresión Génica/fisiología , Hipocampo/crecimiento & desarrollo , MicroARNs/metabolismo , Neurogénesis/fisiología , Neuronas/citología , Transducción de Señal/fisiología , Animales , Proteína de Unión a CREB/fisiología , Diferenciación Celular/fisiología , Línea Celular Tumoral , Citometría de Flujo , Técnicas de Inactivación de Genes , Humanos , Inmunohistoquímica , Ratones , Ratones Transgénicos , MicroARNs/genética , Microscopía ConfocalRESUMEN
Structure-function studies of mammalian and bacterial excitatory amino acid transporters (EAATs), as well as the crystal structure of a related archaeal glutamate transporter, support a model in which TM7, TM8, and the re-entrant loops HP1 and HP2 participate in forming a substrate translocation pathway within each subunit of a trimer. However, the transport mechanism, including precise binding sites for substrates and co-transported ions and changes in the tertiary structure underlying transport, is still not known. In this study, we used chemical cross-linking of introduced cysteine pairs in a cysteine-less version of EAAT1 to examine the dynamics of key domains associated with the translocation pore. Here we show that cysteine substitution at Ala-395, Ala-367, and Ala-440 results in functional single and double cysteine transporters and that in the absence of glutamate or dl-threo-beta-benzyloxyaspartate (dl-TBOA), A395C in the highly conserved TM7 can be cross-linked to A367C in HP1 and to A440C in HP2. The formation of these disulfide bonds is reversible and occurs intra-molecularly. Interestingly, cross-linking A395C to A367C appears to abolish transport, whereas cross-linking A395C to A440C lowers the affinities for glutamate and dl-TBOA but does not change the maximal transport rate. Additionally, glutamate and dl-TBOA binding prevent cross-linking in both double cysteine transporters, whereas sodium binding facilitates cross-linking in the A395C/A367C transporter. These data provide evidence that within each subunit of EAAT1, Ala-395 in TM7 resides close to a residue at the tip of each re-entrant loop (HP1 and HP2) and that these residues are repositioned relative to one another at different steps in the transport cycle. Such behavior likely reflects rearrangements in the tertiary structure of the translocation pore during transport and thus provides constraints for modeling the structural dynamics associated with transport.
Asunto(s)
Transportador 1 de Aminoácidos Excitadores/química , Transportador 1 de Aminoácidos Excitadores/metabolismo , Animales , Transporte Biológico/genética , Células COS , Chlorocebus aethiops , Reactivos de Enlaces Cruzados/metabolismo , Cisteína/genética , Cisteína/metabolismo , Disulfuros/metabolismo , Transportador 1 de Aminoácidos Excitadores/genética , Humanos , Mutagénesis Sitio-Dirigida , Estructura Terciaria de Proteína/genética , Relación Estructura-ActividadRESUMEN
Recent work has shown that cysteine residues introduced into domain 10, a highly hydrophobic segment in the excitatory amino acid transporter 1, react readily when hydrophilic sulfhydryl-modifying reagents are applied extracellularly. To investigate the functional contributions of this region, we mutated each residue in domain 10 (Ala(446)-Gly(459)) to cysteine and assessed the transport kinetics and inhibitor sensitivities of the mutant carriers. Modification of the introduced sulfhydryl group with membrane-impermeant methanethiosulfonate derivatives inhibited substrate transport by all but one functional cysteine mutant. Substrates and/or non-transported inhibitors block thiol modification of most mutants within this region, implying that access to the domain becomes restricted as a consequence of the binding of substrates and substrate analogs. An examination of the temperature dependence of substrate protection for one mutant (I453C) indicates that substrates prevent modification at a step prior to the large conformational changes associated with translocation. When superimposed on a helical model, mutants with similar attributes are positioned in close proximity. Our data are consistent with a model in which domain 10 exists as an alpha-helix at an aqueous interface of the translocation pathway, which can be directly occluded by substrates and inhibitors at an early step in the transport cycle.