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1.
Nat Methods ; 11(3): 338-46, 2014 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-24509633

RESUMEN

Optogenetic tools enable examination of how specific cell types contribute to brain circuit functions. A long-standing question is whether it is possible to independently activate two distinct neural populations in mammalian brain tissue. Such a capability would enable the study of how different synapses or pathways interact to encode information in the brain. Here we describe two channelrhodopsins, Chronos and Chrimson, discovered through sequencing and physiological characterization of opsins from over 100 species of alga. Chrimson's excitation spectrum is red shifted by 45 nm relative to previous channelrhodopsins and can enable experiments in which red light is preferred. We show minimal visual system-mediated behavioral interference when using Chrimson in neurobehavioral studies in Drosophila melanogaster. Chronos has faster kinetics than previous channelrhodopsins yet is effectively more light sensitive. Together these two reagents enable two-color activation of neural spiking and downstream synaptic transmission in independent neural populations without detectable cross-talk in mouse brain slice.


Asunto(s)
Proteínas de Drosophila/metabolismo , Drosophila melanogaster/fisiología , Luz , Neuronas/fisiología , Animales , Proteínas de Drosophila/genética , Datos de Secuencia Molecular , Optogenética , Rodopsina/genética , Rodopsina/metabolismo
3.
J Neurosci ; 30(4): 1463-70, 2010 Jan 27.
Artículo en Inglés | MEDLINE | ID: mdl-20107073

RESUMEN

AMPA receptors (AMPARs) are tetrameric ligand-gated ion channels that couple the energy of glutamate binding to the opening of a transmembrane channel. Crystallographic and electrophysiological analysis of AMPARs has suggested a coupling between (1) cleft closure in the bilobate ligand-binding domain (LBD), (2) the resulting separation of transmembrane helix attachment points across subunit dimers, and (3) agonist efficacy. In general, more efficacious agonists induce greater degrees of cleft closure and transmembrane separation than partial agonists. Several apparent violations of the cleft-closure/efficacy paradigm have emerged, although in all cases, intradimer separation remains as the driving force for channel opening. Here, we examine the structural basis of partial agonism in GluA4 AMPARs. We find that the L651V substitution enhances the relative efficacy of kainate without increasing either LBD cleft closure or transmembrane separation. Instead, the conformational change relative to the wild-type:kainate complex involves a twisting motion with the efficacy contribution opposite from that expected based on previous analyses. As a result, channel opening may involve transmembrane rearrangements with a significant rotational component. Furthermore, a two-dimensional analysis of agonist-induced GluA2 LBD motions suggests that efficacy is not a linearly varying function of lobe 2 displacement vectors, but is rather determined by specific conformational requirements of the transmembrane domains.


Asunto(s)
Agonistas de Aminoácidos Excitadores/química , Agonistas de Aminoácidos Excitadores/farmacología , Ácido Glutámico/metabolismo , Receptores AMPA/agonistas , Receptores AMPA/química , Transmisión Sináptica/fisiología , Sitios de Unión , Línea Celular , Membrana Celular/química , Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Cristalografía por Rayos X , Humanos , Activación del Canal Iónico/efectos de los fármacos , Activación del Canal Iónico/fisiología , Canales Iónicos/agonistas , Canales Iónicos/química , Ácido Kaínico/farmacología , Ligandos , Técnicas de Placa-Clamp , Estructura Terciaria de Proteína/efectos de los fármacos , Estructura Terciaria de Proteína/fisiología , Proteómica , Receptores AMPA/metabolismo
4.
Biochemistry ; 47(52): 13831-41, 2008 Dec 30.
Artículo en Inglés | MEDLINE | ID: mdl-19102704

RESUMEN

AMPA receptors are glutamate-gated ion channels that are essential mediators of synaptic signals in the central nervous system. They form tetramers that are assembled as combinations of subunits GluR1-4, each of which contains a ligand-binding domain (LBD). Crystal structures of the GluR2 LBD have revealed an agonist-binding cleft, which is located between two lobes and which acts like a Venus flytrap. In general, agonist efficacy is correlated with the extent of cleft closure. However, recent observations show that cleft closure is not the sole determinant of the relative efficacy for glutamate receptors. In addition, these studies have focused on the GluR2 subunit, which is the specific target of a physiologically important RNA-editing modification in vivo. We therefore sought to test the generality of the cleft closure-efficacy correlation for other AMPA-R subunits. Here, we present crystal structures of the GluR4(flip) LBD in complex with both full and partial agonists. As for GluR2, both agonists stabilize a closed-cleft conformation, and the partial agonist induces a smaller cleft closure than the full agonist. However, a detailed analysis of LBD-kainate interactions reveals the importance of subtle backbone conformational changes in the ligand-binding pocket in determining the magnitude of agonist-associated conformational changes. Furthermore, the GluR4 subunit exhibits a different correlation between receptor activation and LBD cleft closure than does GluR2.


Asunto(s)
Receptores AMPA/agonistas , Receptores AMPA/química , Animales , Sitios de Unión , Cristalografía por Rayos X , Ligandos , Unión Proteica , Conformación Proteica , Ratas , Receptores AMPA/metabolismo
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