RESUMEN
Neurotransmitter-gated ion channels are allosteric proteins that switch on and off in response to agonist binding. Most studies have focused on the agonist-bound, activated channel while assigning a lesser role to the apo or resting state. Here, we show that nanoscale mobility of resting α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-type ionotropic glutamate receptors (AMPA receptors) predetermines responsiveness to neurotransmitter, allosteric anions and TARP auxiliary subunits. Mobility at rest is regulated by alternative splicing of the flip/flop cassette of the ligand-binding domain, which controls motions in the distant AMPA receptor N-terminal domain (NTD). Flip variants promote moderate NTD movement, which establishes slower channel desensitization and robust regulation by anions and auxiliary subunits. In contrast, greater NTD mobility imparted by the flop cassette acts as a master switch to override allosteric regulation. In AMPA receptor heteromers, TARP stoichiometry further modifies these actions of the flip/flop cassette generating two functionally distinct classes of partially and fully TARPed receptors typical of cerebellar stellate and Purkinje cells.
Asunto(s)
Células de Purkinje/metabolismo , Receptores AMPA/metabolismo , Regulación Alostérica , Sitio Alostérico , Empalme Alternativo , Animales , Cerebelo/citología , Cerebelo/metabolismo , Microscopía por Crioelectrón , Cristalografía por Rayos X , Células HEK293 , Humanos , Activación del Canal Iónico , Proteínas de la Membrana/metabolismo , Proteínas de la Membrana/ultraestructura , Ratones , Microscopía de Fuerza Atómica , Técnicas de Placa-Clamp , Dominios Proteicos , Isoformas de Proteínas/genética , Estructura Cuaternaria de Proteína , Estructura Terciaria de Proteína , Receptores AMPA/genética , Receptores AMPA/ultraestructuraRESUMEN
The ß-secretase (BACE1) initiates processing of the amyloid precursor protein (APP) into Aß peptides, which have been implicated as central players in the pathology of Alzheimer disease. BACE1 has been described as a copper-binding protein and its oligomeric state as being monomeric, dimeric, and/or multimeric, but the native cellular stoichiometry has remained elusive. Here, by using single-molecule fluorescence and in vitro cross-linking experiments with photo-activatable unnatural amino acids, we show that full-length BACE1, independently of its subcellular localization, exists as trimers in human cells. We found that trimerization requires the BACE1 transmembrane sequences (TMSs) and cytoplasmic domains, with residues Ala463 and Cys466 buried within the trimer interface of the sulfur-rich core of the TMSs. Our 3D model predicts that the sulfur-rich core of the trimeric BACE1 TMS is accessible to metal ions, but copper ions did not trigger trimerization. The results of functional assays of endogenous BACE1 suggest that it has a role in intracellular copper compartmentalization by transferring cytosolic copper to intracellular compartments, while leaving the overall cellular copper concentration unaltered. Adding to existing physiological models, our results provide novel insight into the atypical interactions between copper and BACE1 and into its non-enzymatic activities. In conclusion, therapeutic Alzheimer disease prevention strategies aimed at decreasing BACE1 protein levels should be regarded with caution, because adverse effects in copper homeostasis may occur.
Asunto(s)
Secretasas de la Proteína Precursora del Amiloide/metabolismo , Ácido Aspártico Endopeptidasas/metabolismo , Cobre/metabolismo , Citosol/metabolismo , Modelos Moleculares , Alanina/química , Sustitución de Aminoácidos , Secretasas de la Proteína Precursora del Amiloide/antagonistas & inhibidores , Secretasas de la Proteína Precursora del Amiloide/química , Secretasas de la Proteína Precursora del Amiloide/genética , Ácido Aspártico Endopeptidasas/antagonistas & inhibidores , Ácido Aspártico Endopeptidasas/química , Ácido Aspártico Endopeptidasas/genética , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Transporte Biológico , Cisteína/química , Transferencia Resonante de Energía de Fluorescencia , Proteínas Fluorescentes Verdes/química , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Células HEK293 , Humanos , Proteínas Luminiscentes/química , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Microscopía Fluorescente , Mutagénesis Sitio-Dirigida , Mutación Puntual , Conformación Proteica , Pliegue de Proteína , Dominios y Motivos de Interacción de Proteínas , Multimerización de Proteína , Interferencia de ARN , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/metabolismoRESUMEN
Neurotransmitter-gated ion channels adopt different gating modes to fine-tune signaling at central synapses. At glutamatergic synapses, high and low activity of AMPA receptors (AMPARs) is observed when pore-forming subunits coassemble with or without auxiliary subunits, respectively. Whether a common structural pathway accounts for these different gating modes is unclear. Here, we identify two structural motifs that determine the time course of AMPAR channel activation. A network of electrostatic interactions at the apex of the AMPAR ligand-binding domain (LBD) is essential for gating by pore-forming subunits, whereas a conserved motif on the lower, D2 lobe of the LBD prolongs channel activity when auxiliary subunits are present. Accordingly, channel activity is almost entirely abolished by elimination of the electrostatic network but restored via auxiliary protein interactions at the D2 lobe. In summary, we propose that activation of native AMPAR complexes is coordinated by distinct structural pathways, favored by the association/dissociation of auxiliary subunits.
Asunto(s)
Activación del Canal Iónico/fisiología , Potenciales de la Membrana/fisiología , Mutación/fisiología , Receptores AMPA/química , Receptores AMPA/metabolismo , Sitios de Unión/efectos de los fármacos , Sitios de Unión/genética , Cristalografía por Rayos X , Estimulación Eléctrica , Ácido Glutámico/farmacología , Células HEK293 , Humanos , Activación del Canal Iónico/genética , Litio/farmacología , Potenciales de la Membrana/efectos de los fármacos , Modelos Moleculares , Mutación/genética , Técnicas de Placa-Clamp , Estructura Terciaria de Proteína/genética , Estructura Terciaria de Proteína/fisiología , Subunidades de Proteína/genética , Subunidades de Proteína/metabolismo , Receptores AMPA/genética , Electricidad Estática , TransfecciónRESUMEN
KEY POINTS: Kainate receptor heteromerization and auxiliary subunits, Neto1 and Neto2, attenuate polyamine ion-channel block by facilitating blocker permeation. Relief of polyamine block in GluK2/GluK5 heteromers results from a key proline residue that produces architectural changes in the channel pore α-helical region. Auxiliary subunits exert an additive effect to heteromerization, and thus relief of polyamine block is due to a different mechanism. Our findings have broad implications for work on polyamine block of other cation-selective ion channels. ABSTRACT: Channel block and permeation by cytoplasmic polyamines is a common feature of many cation-selective ion channels. Although the channel block mechanism has been studied extensively, polyamine permeation has been considered less significant as it occurs at extreme positive membrane potentials. Here, we show that kainate receptor (KAR) heteromerization and association with auxiliary proteins, Neto1 and Neto2, attenuate polyamine block by enhancing blocker permeation. Consequently, polyamine permeation and unblock occur at more negative and physiologically relevant membrane potentials. In GluK2/GluK5 heteromers, enhanced permeation is due to a single proline residue in GluK5 that alters the dynamics of the α-helical region of the selectivity filter. The effect of auxiliary proteins is additive, and therefore the structural basis of polyamine permeation and unblock is through a different mechanism. As native receptors are thought to assemble as heteromers in complex with auxiliary proteins, our data identify an unappreciated impact of polyamine permeation in shaping the signalling properties of neuronal KARs and point to a structural mechanism that may be shared amongst other cation-selective ion channels.
Asunto(s)
Activación del Canal Iónico , Lipoproteínas LDL/metabolismo , Proteínas de la Membrana/metabolismo , Poliaminas/metabolismo , Receptores de Ácido Kaínico/metabolismo , Animales , Células HEK293 , Humanos , Proteínas Relacionadas con Receptor de LDL , Potenciales de la Membrana , Ratones , Dominios Proteicos , Ratas , Receptores de N-Metil-D-Aspartato , Receptor de Ácido Kaínico GluK2RESUMEN
Desensitization is an important mechanism curtailing the activity of ligand-gated ion channels (LGICs). Although the structural basis of desensitization is not fully resolved, it is thought to be governed by physicochemical properties of bound ligands. Here, we show the importance of an allosteric cation-binding pocket in controlling transitions between activated and desensitized states of rat kainate-type (KAR) ionotropic glutamate receptors (iGluRs). Tethering a positive charge to this pocket sustains KAR activation, preventing desensitization, whereas mutations that disrupt cation binding eliminate channel gating. These different outcomes explain the structural distinction between deactivation and desensitization. Deactivation occurs when the ligand unbinds before the cation, whereas desensitization proceeds if a ligand is bound without cation pocket occupancy. This sequence of events is absent from AMPA-type iGluRs; thus, cations are identified as gatekeepers of KAR gating, a role unique among even closely related LGICs.
Asunto(s)
Receptores de Ácido Kaínico/química , Receptores de Ácido Kaínico/metabolismo , Animales , Sitios de Unión/genética , Células HEK293 , Humanos , Activación del Canal Iónico , Ligandos , Modelos Moleculares , Simulación de Dinámica Molecular , Mutagénesis Sitio-Dirigida , Multimerización de Proteína , Subunidades de Proteína , Ratas , Receptores AMPA/química , Receptores AMPA/genética , Receptores AMPA/metabolismo , Receptores de Ácido Kaínico/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Receptor de Ácido Kaínico GluK2RESUMEN
Kainate-selective ionotropic glutamate receptors (iGluRs) fulfil key roles in the CNS, making them the subject of detailed structural and functional analyses. Although they are known to gate a channel pore with high and low ion-permeation rates, it is still not clear how switches between these gating modes are achieved at the structural level. Here, we uncover an unexpected role for the ligand-binding domain (LBD) dimer assembly in this process. Covalent crosslinking of the dimer interface keeps kainate receptors out of the main open state but permits access to lower conductance states suggesting that significant rearrangements of the dimer interface are required for the receptor to achieve full activation. These observations differ from NMDA-selective iGluRs where constraining dimer movement reduces open-channel probability. In contrast, our data show that restricting movement of the dimer interface interferes with conformational changes that underlie both activation and desensitization. Working within the limits of a common architectural design, we propose functionally diverse iGluR families were able to emerge during evolution by re-deploying existing gating structures to fulfil different tasks.
Asunto(s)
Receptores de Ácido Kaínico/fisiología , Línea Celular , Ácido Glutámico/fisiología , Humanos , Ligandos , Unión Proteica , Multimerización de Proteína , Estructura Terciaria de Proteína , Receptores de Ácido Kaínico/química , Receptor de Ácido Kaínico GluK2RESUMEN
Elucidating subunit stoichiometry of neurotransmitter receptors is preferably carried out in a mammalian expression system where the rules of native protein assembly are strictly obeyed. Although successful in Xenopus oocytes, single subunit counting, manually counting photobleaching steps of GFP-tagged subunits, has been hindered in mammalian cells by high background fluorescence, poor control of expression, and low GFP maturation efficiency. Here, we present a fully automated single-molecule fluorescence counting method that separates tagged proteins on the plasma membrane from background fluorescence and contaminant proteins in the cytosol or the endoplasmic reticulum and determines the protein stoichiometry. Lower GFP maturation rates observed in cells cultured at 37 °C were partly offset using a monomeric version of superfolder GFP. We were able to correctly identify the stoichiometry of GluK2 and α1 glycine receptors. Our approach permits the elucidation of stoichiometry for a wide variety of plasma membrane proteins in mammalian cells with any commercially available TIRF microscope.
Asunto(s)
Membrana Celular/metabolismo , Receptores de Glicina/metabolismo , Receptores de Ácido Kaínico/metabolismo , Animales , Membrana Celular/genética , Fluorescencia , Proteínas Fluorescentes Verdes , Células HEK293 , Humanos , Receptores de Glicina/genética , Receptores de Ácido Kaínico/genética , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Xenopus , Receptor de Ácido Kaínico GluK2RESUMEN
Ca(2+) and/or Zn(2+) entry into neurons and glial cells is often a key step driving the processes of neurodevelopment and disease. As a result, a major pre-occupation of many neuroscientists has been in tracking down when and where nervous tissues express ion channels with appreciable divalent ion permeability. The cobalt (Co(2+))-staining technique is one of the few techniques that allow a snapshot of the entire neuronal circuit, and selectively labels cells expressing divalent-permeable ion channels with a brown-black precipitate. Despite this, its use has been remarkably limited in the past decade. Reluctance to employ this approach has largely been related to an earlier concern with obtaining a reliable and reproducible means of visualizing transported Co(2+). Here we show that recent advances have resolved these issues, opening this straightforward and valuable technique to a much larger neuroscience audience.