RESUMO
Auditory hair cells receive olivocochlear efferent innervation, which refines tonotopic mapping, improves sound discrimination, and mitigates acoustic trauma. The olivocochlear synapse involves α9α10 nicotinic acetylcholine receptors (nAChRs), which assemble in hair cells only coincident with cholinergic innervation and do not express in recombinant mammalian cell lines. Here, genome-wide screening determined that assembly and surface expression of α9α10 require ligand binding. Ion channel function additionally demands an auxiliary subunit, which can be transmembrane inner ear (TMIE) or TMEM132e. Both of these single-pass transmembrane proteins are enriched in hair cells and underlie nonsyndromic human deafness. Inner hair cells from TMIE mutant mice show altered postsynaptic α9α10 function and retain α9α10-mediated transmission beyond the second postnatal week associated with abnormally persistent cholinergic innervation. Collectively, this study provides a mechanism to link cholinergic input with α9α10 assembly, identifies unexpected functions for human deafness genes TMIE/TMEM132e, and enables drug discovery for this elusive nAChR implicated in prevalent auditory disorders.
Assuntos
Surdez/metabolismo , Células Ciliadas Auditivas/metabolismo , Proteínas de Membrana/metabolismo , Receptores Nicotínicos/metabolismo , Animais , Cóclea/metabolismo , Surdez/genética , Humanos , Ligantes , Proteínas de Membrana/genética , Camundongos , Ligação Proteica , Receptores Nicotínicos/genética , Sinapses/metabolismoRESUMO
The α7 nicotinic acetylcholine receptor participates in diverse aspects of brain physiology and disease. Neurons tightly control α7 assembly, which relies upon NACHO, an endoplasmic reticulum (ER)-localized integral membrane protein. By constructing α7 chimeras and mutants, we find that NACHO requires the α7 ectodomain to promote receptor assembly and surface trafficking. Also critical are two amino acids in the α7 second transmembrane domain. NACHO-mediated assembly is independent and separable from that induced by cholinergic ligands or RIC-3 protein, the latter of which acts on the large α7 intracellular loop. Proteomics indicates that NACHO associates with the ER oligosaccharyltransferase machinery and with calnexin. Accordingly, NACHO-mediated effects on α7 assembly and channel function require N-glycosylation and calnexin chaperone activity. These studies identify ER pathways that mediate α7 assembly by NACHO and provide insights into novel pharmacological strategies for these crucial nicotinic receptors.
Assuntos
Retículo Endoplasmático/metabolismo , Proteínas de Membrana/metabolismo , Receptor Nicotínico de Acetilcolina alfa7/metabolismo , Animais , Encéfalo/citologia , Encéfalo/metabolismo , Glicosilação , Células HEK293 , Humanos , Camundongos , Camundongos Knockout , Neurônios/metabolismo , Ratos , Transdução de SinaisRESUMO
Small molecule polyamines are abundant in all life forms and participate in diverse aspects of cell growth and differentiation. Spermidine/spermine acetyltransferase (SAT1) is the rate-limiting enzyme in polyamine catabolism and a primary genetic risk factor for suicidality. Here, using genome-wide screening, we find that SAT1 selectively controls nicotinic acetylcholine receptor (nAChR) biogenesis. SAT1 specifically augments assembly of nAChRs containing α7 or α4ß2, but not α6 subunits. Polyamines are classically studied as regulators of ion channel gating that engage the nAChR channel pore. In contrast, we find polyamine effects on assembly involve the nAChR cytosolic loop. Neurological studies link brain polyamines with neurodegenerative conditions. Our pharmacological and transgenic animal studies find that reducing polyamines enhances cortical neuron nAChR expression and augments nicotine-mediated neuroprotection. Taken together, we describe a most unexpected role for polyamines in regulating ion channel assembly, which provides a new avenue for nAChR neuropharmacology.
Assuntos
Canais Iônicos/metabolismo , Poliaminas/metabolismo , Receptores Nicotínicos/metabolismo , Acetiltransferases , Animais , Biocatálise , DNA Complementar/genética , Elementos Facilitadores Genéticos/genética , Fluorescência , Genoma Humano , Células HEK293 , Humanos , Ativação do Canal Iônico , Camundongos , Neurônios/metabolismo , Neuroproteção/efeitos dos fármacos , Estrutura Secundária de Proteína , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo , Ratos , Receptores Nicotínicos/químicaRESUMO
Vesicle fusion is mediated by assembly of SNARE proteins between opposing membranes. While previous work suggested an active role of SNARE transmembrane domains (TMDs) in promoting membrane merger (Dhara et al., 2016), the underlying mechanism remained elusive. Here, we show that naturally-occurring v-SNARE TMD variants differentially regulate fusion pore dynamics in mouse chromaffin cells, indicating TMD flexibility as a mechanistic determinant that facilitates transmitter release from differentially-sized vesicles. Membrane curvature-promoting phospholipids like lysophosphatidylcholine or oleic acid profoundly alter pore expansion and fully rescue the decelerated fusion kinetics of TMD-rigidifying VAMP2 mutants. Thus, v-SNARE TMDs and phospholipids cooperate in supporting membrane curvature at the fusion pore neck. Oppositely, slowing of pore kinetics by the SNARE-regulator complexin-2 withstands the curvature-driven speeding of fusion, indicating that pore evolution is tightly coupled to progressive SNARE complex formation. Collectively, TMD-mediated support of membrane curvature and SNARE force-generated membrane bending promote fusion pore formation and expansion.
Assuntos
Exocitose , Fusão de Membrana , Complexos Multiproteicos/fisiologia , Neurotransmissores/fisiologia , Fosfolipídeos/metabolismo , Proteínas SNARE/fisiologia , Proteína 2 Associada à Membrana da Vesícula/fisiologia , Animais , Cálcio/fisiologia , Membrana Celular/metabolismo , Células Cultivadas , Células Cromafins , Cinética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas Mutantes/fisiologia , Ligação Proteica , Domínios Proteicos , Vesículas Secretórias/fisiologiaRESUMO
SNAP-25 is an essential component of SNARE complexes driving fast Ca2+-dependent exocytosis. Yet, the functional implications of the tandem-like structure of SNAP-25 are unclear. Here, we have investigated the mechanistic role of the acylated "linker" domain that concatenates the two SNARE motifs within SNAP-25. Refuting older concepts of an inert connector, our detailed structure-function analysis in murine chromaffin cells demonstrates that linker motifs play a crucial role in vesicle priming, triggering, and fusion pore expansion. Mechanistically, we identify two synergistic functions of the SNAP-25 linker: First, linker motifs support t-SNARE interactions and accelerate ternary complex assembly. Second, the acylated N-terminal linker segment engages in local lipid interactions that facilitate fusion triggering and pore evolution, putatively establishing a favorable membrane configuration by shielding phospholipid headgroups and affecting curvature. Hence, the linker is a functional part of the fusion complex that promotes secretion by SNARE interactions as well as concerted lipid interplay.
Assuntos
Células Cromafins/metabolismo , Fosfolipídeos/metabolismo , Vesículas Secretórias/metabolismo , Proteína 25 Associada a Sinaptossoma/metabolismo , Animais , Células Cultivadas , Análise Mutacional de DNA , Feminino , Masculino , Camundongos , Ligação Proteica , Multimerização Proteica , Ratos , Proteínas SNARE/metabolismo , Proteína 25 Associada a Sinaptossoma/genéticaRESUMO
ComplexinII (CpxII) inhibits non-synchronized vesicle fusion, but the underlying mechanisms have remained unclear. Here, we provide evidence that the far C-terminal domain (CTD) of CpxII interferes with SNARE assembly, thereby arresting tonic exocytosis. Acute infusion of a CTD-derived peptide into mouse chromaffin cells enhances synchronous release by diminishing premature vesicle fusion like full-length CpxII, indicating a direct, inhibitory function of the CTD that sets the magnitude of the primed vesicle pool. We describe a high degree of structural similarity between the CpxII CTD and the SNAP25-SN1 domain (C-terminal half) and show that the CTD peptide lowers the rate of SDS-resistant SNARE complex formation in vitro. Moreover, corresponding CpxII:SNAP25 chimeras do restore complexin's function and even 'superclamp' tonic secretion. Collectively, these results support a so far unrecognized clamping mechanism wherein the CpxII C-terminus hinders spontaneous SNARE complex assembly, enabling the build-up of a release-ready pool of vesicles for synchronized Ca2+-triggered exocytosis.
Assuntos
Proteínas Adaptadoras de Transporte Vesicular/química , Exocitose/genética , Proteínas do Tecido Nervoso/química , Vesículas Sinápticas/química , Proteína 25 Associada a Sinaptossoma/química , Proteínas Adaptadoras de Transporte Vesicular/genética , Animais , Cálcio/química , Membrana Celular/química , Membrana Celular/genética , Fusão de Membrana/genética , Camundongos , Proteínas do Tecido Nervoso/genética , Ligação Proteica , Domínios Proteicos/genética , Proteínas SNARE/química , Proteínas SNARE/genética , Vesículas Sinápticas/genética , Proteína 25 Associada a Sinaptossoma/genéticaRESUMO
Vesicle fusion is elementary for intracellular trafficking and release of signal molecules, thus providing the basis for diverse forms of intercellular communication like hormonal regulation or synaptic transmission. A detailed characterization of the mechanisms underlying exocytosis is key to understand how the nervous system integrates information and generates appropriate responses to stimuli. The machinery for vesicular release employs common molecular players in different model systems including neuronal and neuroendocrine cells, in particular members of the SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptors) protein family, Sec1/Munc18-like proteins, and other accessory factors. To achieve temporal precision and speed, excitable cells utilize specialized regulatory proteins like synaptotagmin and complexin, whose interplay putatively synchronizes vesicle fusion and enhances stimulus-secretion coupling. In this review, we aim to highlight recent progress and emerging views on the molecular mechanisms, by which constitutively forming SNAREpins are organized in functional, tightly regulated units for synchronized release. Specifically, we will focus on the role of vesicle associated membrane proteins, also referred to as vesicular SNAREs, in fusion and rapid cargo discharge. We will further discuss the functions of SNARE regulators during exocytosis and focus on chromaffin cell as a model system of choice that allows for detailed structure-function analyses and direct measurements of vesicle fusion under precise control of intracellular [Ca]i.
Assuntos
Células Cromafins/metabolismo , Proteínas SNARE/metabolismo , Animais , Exocitose , Humanos , Fusão de MembranaRESUMO
Vesicle fusion is mediated by an assembly of SNARE proteins between opposing membranes, but it is unknown whether transmembrane domains (TMDs) of SNARE proteins serve mechanistic functions that go beyond passive anchoring of the force-generating SNAREpin to the fusing membranes. Here, we show that conformational flexibility of synaptobrevin-2 TMD is essential for efficient Ca(2+)-triggered exocytosis and actively promotes membrane fusion as well as fusion pore expansion. Specifically, the introduction of helix-stabilizing leucine residues within the TMD region spanning the vesicle's outer leaflet strongly impairs exocytosis and decelerates fusion pore dilation. In contrast, increasing the number of helix-destabilizing, ß-branched valine or isoleucine residues within the TMD restores normal secretion but accelerates fusion pore expansion beyond the rate found for the wildtype protein. These observations provide evidence that the synaptobrevin-2 TMD catalyzes the fusion process by its structural flexibility, actively setting the pace of fusion pore expansion.
Assuntos
Exocitose , Fusão de Membrana , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Vesículas Secretórias/metabolismo , Proteína 2 Associada à Membrana da Vesícula/genética , Proteína 2 Associada à Membrana da Vesícula/metabolismo , Animais , Células Cultivadas , Análise Mutacional de DNA , Camundongos , Modelos Biológicos , Proteínas Mutantes/química , Conformação Proteica , Proteína 2 Associada à Membrana da Vesícula/químicaRESUMO
Despite intensive research, it is still unclear how an immediate and profound acceleration of exocytosis is triggered by appropriate Ca(2+)-stimuli in presynaptic terminals. This is due to the fact that the molecular mechanisms of "docking" and "priming" reactions, which set up secretory vesicles to fuse at millisecond time scale, are extremely hard to study. Yet, driven by a fruitful combination of in vitro and in vivo analyses, our mechanistic understanding of Ca(2+)-triggered vesicle fusion has certainly advanced in the past few years. In this review, we aim to highlight recent progress and emerging views on the molecular mechanisms, by which constitutively forming SNAREpins are organized in functional, tightly regulated units for synchronized release. In particular, we will focus on the role of the small regulatory factor complexin whose function in Ca(2+)-dependent exocytosis has been controversially discussed for more than a decade. Special emphasis will also be laid on the functional relationship of complexin and synaptotagmin, as both proteins possibly act as allies and/or antagonists to govern SNARE-mediated exocytosis.
Assuntos
Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Cálcio/metabolismo , Exocitose , Proteínas do Tecido Nervoso/metabolismo , Vesículas Sinápticas/metabolismo , Humanos , Fusão de Membrana , Modelos Biológicos , Ligação Proteica , Proteínas SNARE/metabolismo , Sinaptotagminas/metabolismoRESUMO
ComplexinII (CpxII) and SynaptotagminI (SytI) have been implicated in regulating the function of SNARE proteins in exocytosis, but their precise mode of action and potential interplay have remained unknown. In this paper, we show that CpxII increases Ca(2+)-triggered vesicle exocytosis and accelerates its secretory rates, providing two independent, but synergistic, functions to enhance synchronous secretion. Specifically, we demonstrate that the C-terminal domain of CpxII increases the pool of primed vesicles by hindering premature exocytosis at submicromolar Ca(2+) concentrations, whereas the N-terminal domain shortens the secretory delay and accelerates the kinetics of Ca(2+)-triggered exocytosis by increasing the Ca(2+) affinity of synchronous secretion. With its C terminus, CpxII attenuates fluctuations of the early fusion pore and slows its expansion but is functionally antagonized by SytI, enabling rapid transmitter discharge from single vesicles. Thus, our results illustrate how key features of CpxII, SytI, and their interplay transform the constitutively active SNARE-mediated fusion mechanism into a highly synchronized, Ca(2+)-triggered release apparatus.
Assuntos
Proteínas Adaptadoras de Transporte Vesicular/fisiologia , Exocitose , Proteínas do Tecido Nervoso/fisiologia , Animais , Sinalização do Cálcio , Células Cultivadas , Células Cromafins/metabolismo , Grânulos Cromafim/metabolismo , Cinética , Fusão de Membrana , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Knockout , Proteínas SNARE/metabolismo , Vesículas Secretórias/metabolismo , Sinaptotagminas/metabolismo , Proteínas de Transporte VesicularRESUMO
Trans-soluble N-ethylmaleimide-sensitive factor attachment protein (SNAP) receptor (SNARE) complexes formed between the SNARE motifs of synaptobrevin II, SNAP-25, and syntaxin play an essential role in Ca(2+)-regulated exocytosis. Apart from the well studied interactions of the SNARE domains, little is known about the functional relevance of other evolutionarily conserved structures in the SNARE proteins. Here, we show that substitution of two highly conserved tryptophan residues within the juxtamembrane domain (JMD) of the vesicular SNARE Synaptobrevin II (SybII) profoundly impairs priming of granules in mouse chromaffin cells without altering catecholamine release from single vesicles. Using molecular dynamic simulations of membrane-embedded SybII, we show that Trp residues of the JMD influence the electrostatic surface potential by controlling the position of neighboring lysine and arginine residues at the membrane-water interface. Our observations indicate a decisive role of the tryptophan moiety of SybII in keeping the vesicles in the release-ready state and support a model wherein tryptophan-mediated protein-lipid interactions assist in bridging the apposing membranes before fusion.
Assuntos
Membrana Celular/metabolismo , Proteínas SNARE/metabolismo , Vesículas Secretórias/metabolismo , Triptofano/metabolismo , Proteína 2 Associada à Membrana da Vesícula/metabolismo , Animais , Células Cultivadas , Exocitose/fisiologia , Camundongos , Camundongos Knockout , Proteínas SNARE/genética , Vesículas Secretórias/genética , Triptofano/genética , Proteína 2 Associada à Membrana da Vesícula/genéticaRESUMO
Parkinson's disease (PD) is a neurodegenerative disorder characterized by motor, cognitive, neuropsychiatric, autonomic, and other nonmotor symptoms. Deep brain stimulation (DBS) at high frequency is now considered the most effective neurosurgical therapy for movement disorders, especially PD. An electrode is chronically implanted in a particular area of the brain and, when continuously stimulated, it significantly alleviates motor symptoms. In Parkinson's disease, the common target nuclei of high frequency stimulation (HFS) are the basal ganglia nuclei, such as the internal segment of the pallidum and the subthalamic nucleus (STN), with a preference for the STN in recent years. Two fundamental mechanisms have been proposed to underlie the beneficial effects of HFS: either silencing or excitation of STN neurons. This article highlights the recent views concerned with the mechanisms of DBS. Although the efficacy of DBS for the motor symptoms of advanced PD is well established, the effects of DBS on the cognitive and neuropsychiatric symptoms are less clear. The cognitive aspects of DBS for PD have recently been of considerable clinical and pathophysiological interest. This article also reviews the published literature on the cognitive aspects of DBS for PD.