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1.
Nature ; 614(7946): 153-159, 2023 02.
Artigo em Inglês | MEDLINE | ID: mdl-36697829

RESUMO

Mitochondria have crucial roles in cellular energetics, metabolism, signalling and quality control1-4. They contain around 1,000 different proteins that often assemble into complexes and supercomplexes such as respiratory complexes and preprotein translocases1,3-7. The composition of the mitochondrial proteome has been characterized1,3,5,6; however, the organization of mitochondrial proteins into stable and dynamic assemblies is poorly understood for major parts of the proteome1,4,7. Here we report quantitative mapping of mitochondrial protein assemblies using high-resolution complexome profiling of more than 90% of the yeast mitochondrial proteome, termed MitCOM. An analysis of the MitCOM dataset resolves >5,200 protein peaks with an average of six peaks per protein and demonstrates a notable complexity of mitochondrial protein assemblies with distinct appearance for respiration, metabolism, biogenesis, dynamics, regulation and redox processes. We detect interactors of the mitochondrial receptor for cytosolic ribosomes, of prohibitin scaffolds and of respiratory complexes. The identification of quality-control factors operating at the mitochondrial protein entry gate reveals pathways for preprotein ubiquitylation, deubiquitylation and degradation. Interactions between the peptidyl-tRNA hydrolase Pth2 and the entry gate led to the elucidation of a constitutive pathway for the removal of preproteins. The MitCOM dataset-which is accessible through an interactive profile viewer-is a comprehensive resource for the identification, organization and interaction of mitochondrial machineries and pathways.


Assuntos
Proteínas Fúngicas , Mitocôndrias , Proteínas Mitocondriais , Transporte Proteico , Proteoma , Saccharomyces cerevisiae , Proteínas de Transporte/metabolismo , Mitocôndrias/metabolismo , Proteínas Mitocondriais/metabolismo , Proteoma/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas Fúngicas/metabolismo , Respiração Celular , Ribossomos , Conjuntos de Dados como Assunto
2.
Circ Res ; 134(4): 346-350, 2024 02 16.
Artigo em Inglês | MEDLINE | ID: mdl-38359093

RESUMO

Transient receptor potential (TRP) cation channels are a diverse family of channels whose members play prominent roles as cellular sensors and effectors. The important role of TRP channels (and mechanosensitive piezo channels) in the complex interaction of our senses with the environment was underlined by the award of the Nobel Prize in Physiology or Medicine to 2 pioneers in this field, David Julius and Ardem Patapoutian. There are many competent and comprehensive reviews on many aspects of the TRP channels, and there is no intention to expand on them. Rather, after an introduction to the nomenclature, the molecular architecture of native TRP channel/protein complexes in vivo will be summarized using TRP channels of the canonical transient receptor potential subfamily as an example. This molecular architecture provides the basis for the signatures of native canonical transient receptor potential currents and their control by endogenous modulators and potential drugs.


Assuntos
Canais de Potencial de Receptor Transitório
3.
EMBO Rep ; 25(6): 2610-2634, 2024 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-38698221

RESUMO

GABAB receptors (GBRs), the G protein-coupled receptors for GABA, regulate synaptic transmission throughout the brain. A main synaptic function of GBRs is the gating of Cav2.2-type Ca2+ channels. However, the cellular compartment where stable GBR/Cav2.2 signaling complexes form remains unknown. In this study, we demonstrate that the vesicular protein synaptotagmin-11 (Syt11) binds to both the auxiliary GBR subunit KCTD16 and Cav2.2 channels. Through these dual interactions, Syt11 recruits GBRs and Cav2.2 channels to post-Golgi vesicles, thus facilitating assembly of GBR/Cav2.2 signaling complexes. In addition, Syt11 stabilizes GBRs and Cav2.2 channels at the neuronal plasma membrane by inhibiting constitutive internalization. Neurons of Syt11 knockout mice exhibit deficits in presynaptic GBRs and Cav2.2 channels, reduced neurotransmitter release, and decreased GBR-mediated presynaptic inhibition, highlighting the critical role of Syt11 in the assembly and stable expression of GBR/Cav2.2 complexes. These findings support that Syt11 acts as a vesicular scaffold protein, aiding in the assembly of signaling complexes from low-abundance components within transport vesicles. This mechanism enables insertion of pre-assembled functional signaling units into the synaptic membrane.


Assuntos
Camundongos Knockout , Transdução de Sinais , Sinaptotagminas , Animais , Sinaptotagminas/metabolismo , Sinaptotagminas/genética , Camundongos , Humanos , Neurônios/metabolismo , Transmissão Sináptica , Receptores de GABA-B/metabolismo , Receptores de GABA-B/genética , Terminações Pré-Sinápticas/metabolismo , Canais de Cálcio Tipo N/metabolismo , Canais de Cálcio Tipo N/genética , Complexo de Golgi/metabolismo , Ligação Proteica , Células HEK293
4.
J Physiol ; 599(10): 2639-2653, 2021 05.
Artigo em Inglês | MEDLINE | ID: mdl-32749711

RESUMO

AMPA-type glutamate receptors (AMPARs), the key elements of fast excitatory neurotransmission in the brain, are receptor ion channels whose core is assembled from pore-forming and three distinct types of auxiliary subunits. While it is well established that this assembly occurs in the endoplasmic reticulum (ER), it has remained largely enigmatic how this receptor-building happens. Here we review recent findings on the biogenesis of AMPARs in native neurons as a multistep production line that is defined and operated by distinct ER-resident helper proteins, and we discuss how impairment of these operators by mutations or targeted gene-inactivation leads to severe phenotypes in both humans and rodents. We suggest that the recent data on AMPAR biogenesis provide new insights into a process that is key to the formation and operation of excitatory synapses and their activity-dependent dynamics, as well as for the operation of the mammalian brain under normal and pathological conditions.


Assuntos
Ácido Glutâmico , Receptores de AMPA , Retículo Endoplasmático/metabolismo , Receptores de AMPA/metabolismo , Transmissão Sináptica , Ácido alfa-Amino-3-hidroxi-5-metil-4-isoxazol Propiônico
5.
EMBO J ; 36(18): 2770-2789, 2017 09 15.
Artigo em Inglês | MEDLINE | ID: mdl-28790178

RESUMO

Canonical transient receptor potential (TRPC) channels influence various neuronal functions. Using quantitative high-resolution mass spectrometry, we demonstrate that TRPC1, TRPC4, and TRPC5 assemble into heteromultimers with each other, but not with other TRP family members in the mouse brain and hippocampus. In hippocampal neurons from Trpc1/Trpc4/Trpc5-triple-knockout (Trpc1/4/5-/-) mice, lacking any TRPC1-, TRPC4-, or TRPC5-containing channels, action potential-triggered excitatory postsynaptic currents (EPSCs) were significantly reduced, whereas frequency, amplitude, and kinetics of quantal miniature EPSC signaling remained unchanged. Likewise, evoked postsynaptic responses in hippocampal slice recordings and transient potentiation after tetanic stimulation were decreased. In vivo, Trpc1/4/5-/- mice displayed impaired cross-frequency coupling in hippocampal networks and deficits in spatial working memory, while spatial reference memory was unaltered. Trpc1/4/5-/- animals also exhibited deficiencies in adapting to a new challenge in a relearning task. Our results indicate the contribution of heteromultimeric channels from TRPC1, TRPC4, and TRPC5 subunits to the regulation of mechanisms underlying spatial working memory and flexible relearning by facilitating proper synaptic transmission in hippocampal neurons.


Assuntos
Hipocampo/fisiologia , Memória de Curto Prazo , Multimerização Proteica , Transmissão Sináptica , Canais de Cátion TRPC/metabolismo , Animais , Técnicas de Inativação de Genes , Hipocampo/metabolismo , Espectrometria de Massas , Camundongos , Camundongos Knockout , Canais de Cátion TRPC/genética
6.
Proc Natl Acad Sci U S A ; 114(22): 5707-5712, 2017 05 30.
Artigo em Inglês | MEDLINE | ID: mdl-28507132

RESUMO

Voltage-activated calcium (Cav) channels couple intracellular signaling pathways to membrane potential by providing Ca2+ ions as second messengers at sufficiently high concentrations to modulate effector proteins located in the intimate vicinity of those channels. Here we show that protein kinase Cß (PKCß) and brain nitric oxide synthase (NOS1), both identified by proteomic analysis as constituents of the protein nano-environment of Cav2 channels in the brain, directly coassemble with Cav2.2 channels upon heterologous coexpression. Within Cav2.2-PKCß and Cav2.2-NOS1 complexes voltage-triggered Ca2+ influx through the Cav channels reliably initiates enzymatic activity within milliseconds. Using BKCa channels as target sensors for nitric oxide and protein phosphorylation together with high concentrations of Ca2+ buffers showed that the complex-mediated Ca2+ signaling occurs in local signaling domains at the plasma membrane. Our results establish Cav2-enzyme complexes as molecular entities for fast electrochemical coupling that reliably convert brief membrane depolarization into precisely timed intracellular signaling events in the mammalian brain.


Assuntos
Canais de Cálcio Tipo N/metabolismo , Sinalização do Cálcio/fisiologia , Potenciais da Membrana/fisiologia , Óxido Nítrico Sintase Tipo I/metabolismo , Proteína Quinase C beta/metabolismo , Animais , Células CHO , Cálcio/metabolismo , Linhagem Celular , Membrana Celular/metabolismo , Cricetulus , Complexos Multiproteicos/metabolismo , Técnicas de Patch-Clamp
7.
Proc Natl Acad Sci U S A ; 114(7): E1253-E1262, 2017 02 14.
Artigo em Inglês | MEDLINE | ID: mdl-28154140

RESUMO

Establishment, specification, and validation of synaptic connections are thought to be mediated by interactions between pre- and postsynaptic cell-adhesion molecules. Arguably, the best-characterized transsynaptic interactions are formed by presynaptic neurexins, which bind to diverse postsynaptic ligands. In a proteomic screen of neurexin-1 (Nrxn1) complexes immunoisolated from mouse brain, we identified carbonic anhydrase-related proteins CA10 and CA11, two homologous, secreted glycoproteins of unknown function that are predominantly expressed in brain. We found that CA10 directly binds in a cis configuration to a conserved membrane-proximal, extracellular sequence of α- and ß-neurexins. The CA10-neurexin complex is stable and stoichiometric, and results in formation of intermolecular disulfide bonds between conserved cysteine residues in neurexins and CA10. CA10 promotes surface expression of α- and ß-neurexins, suggesting that CA10 may form a complex with neurexins in the secretory pathway that facilitates surface transport of neurexins. Moreover, we observed that the Nrxn1 gene expresses from an internal 3' promoter a third isoform, Nrxn1γ, that lacks all Nrxn1 extracellular domains except for the membrane-proximal sequences and that also tightly binds to CA10. Our data expand the understanding of neurexin-based transsynaptic interaction networks by providing further insight into the interactions nucleated by neurexins at the synapse.


Assuntos
Encéfalo/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Moléculas de Adesão de Célula Nervosa/metabolismo , Neurônios/metabolismo , Sequência de Aminoácidos , Animais , Proteínas de Ligação ao Cálcio , Sequência Conservada , Células HEK293 , Humanos , Ligantes , Camundongos
8.
Cereb Cortex ; 27(3): 2318-2334, 2017 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-27073217

RESUMO

Cholecystokinin-expressing interneurons (CCK-INs) mediate behavior state-dependent inhibition in cortical circuits and themselves receive strong GABAergic input. However, it remains unclear to what extent GABAB receptors (GABABRs) contribute to their inhibitory control. Using immunoelectron microscopy, we found that CCK-INs in the rat hippocampus possessed high levels of dendritic GABABRs and KCTD12 auxiliary proteins, whereas postsynaptic effector Kir3 channels were present at lower levels. Consistently, whole-cell recordings revealed slow GABABR-mediated inhibitory postsynaptic currents (IPSCs) in most CCK-INs. In spite of the higher surface density of GABABRs in CCK-INs than in CA1 principal cells, the amplitudes of IPSCs were comparable, suggesting that the expression of Kir3 channels is the limiting factor for the GABABR currents in these INs. Morphological analysis showed that CCK-INs were diverse, comprising perisomatic-targeting basket cells (BCs), as well as dendrite-targeting (DT) interneurons, including a previously undescribed DT type. GABABR-mediated IPSCs in CCK-INs were large in BCs, but small in DT subtypes. In response to prolonged activation, GABABR-mediated currents displayed strong desensitization, which was absent in KCTD12-deficient mice. This study highlights that GABABRs differentially control CCK-IN subtypes, and the kinetics and desensitization of GABABR-mediated currents are modulated by KCTD12 proteins.


Assuntos
Colecistocinina/metabolismo , Canais de Potássio Corretores do Fluxo de Internalização Acoplados a Proteínas G/metabolismo , Potenciais Pós-Sinápticos Inibidores/fisiologia , Interneurônios/metabolismo , Canais de Potássio/metabolismo , Receptores de GABA-A/metabolismo , Animais , Região CA1 Hipocampal/metabolismo , Região CA1 Hipocampal/ultraestrutura , Dendritos/metabolismo , Dendritos/ultraestrutura , Imuno-Histoquímica , Interneurônios/ultraestrutura , Masculino , Microscopia Imunoeletrônica , Técnicas de Patch-Clamp , Ratos Wistar , Técnicas de Cultura de Tecidos
9.
Mol Cell Proteomics ; 15(2): 669-81, 2016 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-26598645

RESUMO

Blue native (BN) gel electrophoresis is a powerful method for protein separation. Combined with liquid chromatography-tandem mass spectrometry (LC-MS/MS), it enables large scale identification of protein complexes and their subunits. Current BN-MS approaches, however, are limited in size resolution, comprehensiveness, and quantification. Here, we present a new methodology combining defined sub-millimeter slicing of BN gels by a cryo-microtome with high performance LC-MS/MS and label-free quantification of protein amounts. Application of this cryo-slicing BN-MS approach to mitochondria from rat brain demonstrated a high degree of comprehensiveness, accuracy, and size resolution. The technique provided abundance-mass profiles for 774 mitochondrial proteins, including all canonical subunits of the oxidative respiratory chain assembled into 13 distinct (super-)complexes. Moreover, the data revealed COX7R as a constitutive subunit of distinct super-complexes and identified novel assemblies of voltage-dependent anion channels/porins and TOM proteins. Together, cryo-slicing BN-MS enables quantitative profiling of complexomes with resolution close to the limits of native gel electrophoresis.


Assuntos
Cromatografia Líquida/métodos , Espectrometria de Massas/métodos , Proteínas Mitocondriais/biossíntese , Biossíntese de Proteínas/genética , Animais , Encéfalo/metabolismo , Transporte de Elétrons/genética , Eletroforese em Gel Bidimensional , Mitocôndrias/metabolismo , Proteínas Mitocondriais/genética , Ratos , Espectrometria de Massas em Tandem/métodos
10.
Physiol Rev ; 90(4): 1437-59, 2010 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-20959620

RESUMO

Molecular research on ion channels has demonstrated that many of these integral membrane proteins associate with partner proteins, often versatile in their function, or even assemble into stable macromolecular complexes that ensure specificity and proper rate of the channel-mediated signal transduction. Calcium-activated potassium (K(Ca)) channels that link excitability and intracellular calcium concentration are responsible for a wide variety of cellular processes ranging from regulation of smooth muscle tone to modulation of neurotransmission and control of neuronal firing pattern. Most of these functions are brought about by interaction of the channels' pore-forming subunits with distinct partner proteins. In this review we summarize recent insights into protein complexes associated with K(Ca) channels as revealed by proteomic research and discuss the results available on structure and function of these complexes and on the underlying protein-protein interactions. Finally, the results are related to their significance for the function of K(Ca) channels under cellular conditions.


Assuntos
Complexos Multiproteicos/fisiologia , Canais de Potássio Cálcio-Ativados/fisiologia , Animais , Regulação da Expressão Gênica/fisiologia , Canais de Potássio Ativados por Cálcio de Condutância Alta/fisiologia , Modelos Moleculares , Conformação Proteica , Transporte Proteico , Proteômica , Canais de Potássio Ativados por Cálcio de Condutância Baixa/fisiologia
11.
Nature ; 465(7295): 231-5, 2010 May 13.
Artigo em Inglês | MEDLINE | ID: mdl-20400944

RESUMO

GABA(B) receptors are the G-protein-coupled receptors for gamma-aminobutyric acid (GABA), the main inhibitory neurotransmitter in the brain. They are expressed in almost all neurons of the brain, where they regulate synaptic transmission and signal propagation by controlling the activity of voltage-gated calcium (Ca(v)) and inward-rectifier potassium (K(ir)) channels. Molecular cloning revealed that functional GABA(B) receptors are formed by the heteromeric assembly of GABA(B1) with GABA(B2) subunits. However, cloned GABA(B(1,2)) receptors failed to reproduce the functional diversity observed with native GABA(B) receptors. Here we show by functional proteomics that GABA(B) receptors in the brain are high-molecular-mass complexes of GABA(B1), GABA(B2) and members of a subfamily of the KCTD (potassium channel tetramerization domain-containing) proteins. KCTD proteins 8, 12, 12b and 16 show distinct expression profiles in the brain and associate tightly with the carboxy terminus of GABA(B2) as tetramers. This co-assembly changes the properties of the GABA(B(1,2)) core receptor: the KCTD proteins increase agonist potency and markedly alter the G-protein signalling of the receptors by accelerating onset and promoting desensitization in a KCTD-subtype-specific manner. Taken together, our results establish the KCTD proteins as auxiliary subunits of GABA(B) receptors that determine the pharmacology and kinetics of the receptor response.


Assuntos
Complexos Multiproteicos/química , Complexos Multiproteicos/metabolismo , Multimerização Proteica , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo , Receptores de GABA-B/química , Receptores de GABA-B/metabolismo , Animais , Células CHO , Cricetinae , Cricetulus , Condutividade Elétrica , Agonistas dos Receptores de GABA-B , Proteínas Heterotriméricas de Ligação ao GTP/metabolismo , Cinética , Camundongos , Neurônios/metabolismo , Oócitos/metabolismo , Potássio/metabolismo , Canais de Potássio/metabolismo , Estrutura Terciária de Proteína , Ratos , Ratos Wistar , Transdução de Sinais , Xenopus
12.
J Neurosci ; 34(46): 15159-69, 2014 Nov 12.
Artigo em Inglês | MEDLINE | ID: mdl-25392484

RESUMO

Voltage- and ligand-gated ion channels form the molecular basis of cellular excitability. With >400 members and accounting for ∼1.5% of the human genome, ion channels are some of the most well studied of all proteins in heterologous expression systems. Yet, ion channels often exhibit unexpected properties in vivo because of their interaction with a variety of signaling/scaffolding proteins. Such interactions can influence the function and localization of ion channels, as well as their coupling to intracellular second messengers and pathways, thus increasing the signaling potential of these ion channels in neurons. Moreover, functions have been ascribed to ion channels that are largely independent of their ion-conducting roles. Molecular and functional dissection of the ion channel proteome/interactome has yielded new insights into the composition of ion channel complexes and how their dysregulation leads to human disease.


Assuntos
Canais Iônicos/fisiologia , Proteômica , Transdução de Sinais/fisiologia , Animais , Adesão Celular/fisiologia , Humanos , Ativação do Canal Iônico/fisiologia , Neurônios/fisiologia , Subunidades Proteicas/fisiologia
13.
J Neurosci ; 34(41): 13586-99, 2014 Oct 08.
Artigo em Inglês | MEDLINE | ID: mdl-25297088

RESUMO

Parkinson disease (PD) is an α-synucleinopathy resulting in the preferential loss of highly vulnerable dopamine (DA) substantia nigra (SN) neurons. Mutations (e.g., A53T) in the α-synuclein gene (SNCA) are sufficient to cause PD, but the mechanism of their selective action on vulnerable DA SN neurons is unknown. In a mouse model overexpressing mutant α-synuclein (A53T-SNCA), we identified a SN-selective increase of in vivo firing frequencies in DA midbrain neurons, which was not observed in DA neurons in the ventral tegmental area. The selective and age-dependent gain-of-function phenotype of A53T-SCNA overexpressing DA SN neurons was in part mediated by an increase of their intrinsic pacemaker frequency caused by a redox-dependent impairment of A-type Kv4.3 potassium channels. This selective enhancement of "stressful pacemaking" of DA SN neurons in vivo defines a functional response to mutant α-synuclein that might be useful as a novel biomarker for the "DA system at risk" before the onset of neurodegeneration in PD.


Assuntos
Neurônios Dopaminérgicos/fisiologia , Mutação/fisiologia , Estresse Oxidativo/fisiologia , Canais de Potássio Shal/fisiologia , Substância Negra/fisiologia , alfa-Sinucleína/genética , Envelhecimento/fisiologia , Animais , Fenômenos Eletrofisiológicos , Glutationa/metabolismo , Glutationa/fisiologia , Ativação do Canal Iônico/fisiologia , Masculino , Camundongos , Mutação/genética , Substância Negra/citologia , Substância Negra/crescimento & desenvolvimento , Área Tegmentar Ventral/crescimento & desenvolvimento , Área Tegmentar Ventral/fisiologia
14.
EMBO J ; 30(14): 2793-804, 2011 Jun 24.
Artigo em Inglês | MEDLINE | ID: mdl-21701557

RESUMO

Prestin, a transporter-like protein of the SLC26A family, acts as a piezoelectric transducer that mediates the fast electromotility of outer hair cells required for cochlear amplification and auditory acuity in mammals. Non-mammalian prestin orthologues are anion transporters without piezoelectric activity. Here, we generated synthetic prestin (SynPres), a chimera of mammalian and non-mammalian prestin exhibiting both, piezoelectric properties and anion transport. SynPres delineates two distinct domains in the protein's transmembrane core that are necessary and sufficient for generating electromotility and associated non-linear charge movement (NLC). Functional analysis of SynPres showed that the amplitude of NLC and hence electromotility are determined by the transport of monovalent anions. Thus, prestin-mediated electromotility is a dual-step process: transport of anions by an alternate access cycle, followed by an anion-dependent transition generating electromotility. The findings define structural and functional determinants of prestin's piezoelectric activity and indicate that the electromechanical process evolved from the ancestral transport mechanism.


Assuntos
Proteínas de Transporte de Ânions/metabolismo , Membrana Celular/metabolismo , Movimento Celular , Capacitância Elétrica , Células Ciliadas Auditivas Externas/fisiologia , Proteínas Motores Moleculares/fisiologia , Proteínas de Peixe-Zebra/metabolismo , Animais , Proteínas de Transporte de Ânions/química , Proteínas de Transporte de Ânions/genética , Ânions/metabolismo , Células CHO , Cricetinae , Cricetulus , Eletrofisiologia , Transporte de Íons , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas Motores Moleculares/química , Técnicas de Cultura de Órgãos , Estrutura Terciária de Proteína , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Transportadores de Sulfato , Proteínas de Peixe-Zebra/química , Proteínas de Peixe-Zebra/genética
15.
J Neurosci ; 33(17): 7358-67, 2013 Apr 24.
Artigo em Inglês | MEDLINE | ID: mdl-23616542

RESUMO

Large conductance Ca(2+)- and voltage-activated potassium channels (BKCa) shape neuronal excitability and signal transduction. This reflects the integrated influences of transmembrane voltage and intracellular calcium concentration ([Ca(2+)]i) that gate the channels. This dual gating has been mainly studied as voltage-triggered gating modulated by defined steady-state [Ca(2+)]i, a paradigm that does not approximate native conditions. Here we use submillisecond changes of [Ca(2+)]i to investigate the time course of the Ca(2+)-triggered gating of BKCa channels expressed in Chinese hamster ovary cells at distinct membrane potentials in the physiological range. The results show that Ca(2+) can effectively gate BKCa channels and that Ca(2+) gating is largely different from voltage-driven gating. Most prominently, Ca(2+) gating displays a pronounced delay in the millisecond range between Ca(2+) application and channel opening (pre-onset delay) and exhibits slower kinetics across the entire [Ca(2+)]i-voltage plane. Both characteristics are selectively altered by co-assembled BKß4 or an epilepsy-causing mutation that either slows deactivation or speeds activation and reduces the pre-onset delay, respectively. Similarly, co-assembly of the BKCa channels with voltage-activated Ca(2+) (Cav) channels, mirroring the native configuration, decreased the pre-onset delay to submillisecond values. In BKCa-Cav complexes, the time course of the hyperpolarizing K(+)-current response is dictated by the Ca(2+) gating of the BKCa channels. Consistent with Cav-mediated Ca(2+) influx, gating was fastest at hyperpolarized potentials, but decreased with depolarization of the membrane potential. Our results demonstrate that under experimental paradigms meant to approximate the physiological conditions BKCa channels primarily operate as ligand-activated channels gated by intracellular Ca(2+) and that Ca(2+) gating is tuned for fast responses in neuronal BKCa-Cav complexes.


Assuntos
Cálcio/metabolismo , Ativação do Canal Iônico/fisiologia , Canais de Potássio Ativados por Cálcio de Condutância Alta/fisiologia , Animais , Células CHO , Cálcio/fisiologia , Cricetinae , Cricetulus , Canais de Potássio Ativados por Cálcio de Condutância Alta/metabolismo , Ligantes
16.
J Neurosci ; 33(22): 9508-19, 2013 May 29.
Artigo em Inglês | MEDLINE | ID: mdl-23719817

RESUMO

The encoding of auditory information with indefatigable precision requires efficient resupply of vesicles at inner hair cell (IHC) ribbon synapses. Otoferlin, a transmembrane protein responsible for deafness in DFNB9 families, has been postulated to act as a calcium sensor for exocytosis as well as to be involved in rapid vesicle replenishment of IHCs. However, the molecular basis of vesicle recycling in IHCs is largely unknown. In the present study, we used high-resolution liquid chromatography coupled with mass spectrometry to copurify otoferlin interaction partners in the mammalian cochlea. We identified multiple subunits of the adaptor protein complex AP-2 (CLAP), an essential component of clathrin-mediated endocytosis, as binding partners of otoferlin in rats and mice. The interaction between otoferlin and AP-2 was confirmed by coimmunoprecipitation. We also found that AP-2 interacts with myosin VI, another otoferlin binding partner important for clathrin-mediated endocytosis (CME). The expression of AP-2 in IHCs was verified by reverse transcription PCR. Confocal microscopy experiments revealed that the expression of AP-2 and its colocalization with otoferlin is confined to mature IHCs. When CME was inhibited by blocking dynamin action, real-time changes in membrane capacitance showed impaired synaptic vesicle replenishment in mature but not immature IHCs. We suggest that an otoferlin-AP-2 interaction drives Ca(2+)- and stimulus-dependent compensating CME in mature IHCs.


Assuntos
Clatrina/fisiologia , Cóclea/fisiologia , Endocitose/fisiologia , Células Ciliadas Auditivas Internas/fisiologia , Proteínas de Membrana/fisiologia , Complexo 2 de Proteínas Adaptadoras/fisiologia , Animais , Membrana Celular/fisiologia , Cóclea/citologia , Fenômenos Eletrofisiológicos , Imuno-Histoquímica , Imunoprecipitação , Espectrometria de Massas , Camundongos , Microscopia Confocal , Cadeias Pesadas de Miosina/fisiologia , Ligação Proteica , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Ratos , Ratos Wistar , Reação em Cadeia da Polimerase em Tempo Real , Sinapses/fisiologia
17.
J Biol Chem ; 288(34): 24848-56, 2013 Aug 23.
Artigo em Inglês | MEDLINE | ID: mdl-23843457

RESUMO

GABA(B) receptors are the G-protein coupled receptors (GPCRs) for GABA, the main inhibitory neurotransmitter in the central nervous system. Native GABA(B) receptors comprise principle and auxiliary subunits that regulate receptor properties in distinct ways. The principle subunits GABA(B1a), GABA(B1b), and GABA(B2) form fully functional heteromeric GABA(B(1a,2)) and GABA(B(1b,2)) receptors. Principal subunits regulate forward trafficking of the receptors from the endoplasmic reticulum to the plasma membrane and control receptor distribution to axons and dendrites. The auxiliary subunits KCTD8, -12, -12b, and -16 are cytosolic proteins that influence agonist potency and G-protein signaling of GABA(B(1a,2)) and GABA(B(1b,2)) receptors. Here, we used transfected cells to study assembly, surface trafficking, and internalization of GABA(B) receptors in the presence of the KCTD12 subunit. Using bimolecular fluorescence complementation and metabolic labeling, we show that GABA(B) receptors associate with KCTD12 while they reside in the endoplasmic reticulum. Glycosylation experiments support that association with KCTD12 does not influence maturation of the receptor complex. Immunoprecipitation and bioluminescence resonance energy transfer experiments demonstrate that KCTD12 remains associated with the receptor during receptor activity and receptor internalization from the cell surface. We further show that KCTD12 reduces constitutive receptor internalization and thereby increases the magnitude of receptor signaling at the cell surface. Accordingly, knock-out or knockdown of KCTD12 in cultured hippocampal neurons reduces the magnitude of the GABA(B) receptor-mediated K(+) current response. In summary, our experiments support that the up-regulation of functional GABA(B) receptors at the neuronal plasma membrane is an additional physiological role of the auxiliary subunit KCTD12.


Assuntos
Hipocampo/metabolismo , Neurônios/metabolismo , Canais de Potássio/metabolismo , Potássio/metabolismo , Multimerização Proteica/fisiologia , Receptores de GABA-B/metabolismo , Transdução de Sinais/fisiologia , Animais , Células COS , Membrana Celular/genética , Membrana Celular/metabolismo , Chlorocebus aethiops , Hipocampo/citologia , Camundongos , Camundongos Knockout , Neurônios/citologia , Canais de Potássio/genética , Receptores de GABA-B/genética
18.
Am J Hum Genet ; 88(4): 422-32, 2011 Apr 08.
Artigo em Inglês | MEDLINE | ID: mdl-21419380

RESUMO

Megalencephalic leukoencephalopathy with subcortical cysts (MLC) is a leukodystrophy characterized by early-onset macrocephaly and delayed-onset neurological deterioration. Recessive MLC1 mutations are observed in 75% of patients with MLC. Genetic-linkage studies failed to identify another gene. We recently showed that some patients without MLC1 mutations display the classical phenotype; others improve or become normal but retain macrocephaly. To find another MLC-related gene, we used quantitative proteomic analysis of affinity-purified MLC1 as an alternative approach and found that GlialCAM, an IgG-like cell adhesion molecule that is also called HepaCAM and is encoded by HEPACAM, is a direct MLC1-binding partner. Analysis of 40 MLC patients without MLC1 mutations revealed multiple different HEPACAM mutations. Ten patients with the classical, deteriorating phenotype had two mutations, and 18 patients with the improving phenotype had one mutation. Most parents with a single mutation had macrocephaly, indicating dominant inheritance. In some families with dominant HEPACAM mutations, the clinical picture and magnetic resonance imaging normalized, indicating that HEPACAM mutations can cause benign familial macrocephaly. In other families with dominant HEPACAM mutations, patients had macrocephaly and mental retardation with or without autism. Further experiments demonstrated that GlialCAM and MLC1 both localize in axons and colocalize in junctions between astrocytes. GlialCAM is additionally located in myelin. Mutant GlialCAM disrupts the localization of MLC1-GlialCAM complexes in astrocytic junctions in a manner reflecting the mode of inheritance. In conclusion, GlialCAM is required for proper localization of MLC1. HEPACAM is the second gene found to be mutated in MLC. Dominant HEPACAM mutations can cause either macrocephaly and mental retardation with or without autism or benign familial macrocephaly.


Assuntos
Transtorno Autístico/genética , Moléculas de Adesão Celular Neuronais/genética , Deficiência Intelectual/genética , Megalencefalia/genética , Mutação , Proteínas/genética , Sequência de Aminoácidos , Animais , Transtorno Autístico/metabolismo , Encéfalo/metabolismo , Moléculas de Adesão Celular Neuronais/metabolismo , Proteínas de Ciclo Celular , Células Cultivadas , Cistos/genética , Cistos/metabolismo , Genes Dominantes , Doenças Desmielinizantes Hereditárias do Sistema Nervoso Central/genética , Doenças Desmielinizantes Hereditárias do Sistema Nervoso Central/metabolismo , Humanos , Deficiência Intelectual/metabolismo , Megalencefalia/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Dados de Sequência Molecular , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas/genética , Proteínas/metabolismo , Ratos , Homologia de Sequência de Aminoácidos
19.
Mol Cell Proteomics ; 11(2): M111.007955, 2012 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-22067099

RESUMO

Affinity purification (AP) of protein complexes combined with LC-MS/MS analysis is the current method of choice for identification of protein-protein interactions. Their interpretation with respect to significance, specificity, and selectivity requires quantification methods coping with enrichment factors of more than 1000-fold, variable amounts of total protein, and low abundant, unlabeled samples. We used standardized samples (0.1-1000 fmol) measured on high resolution hybrid linear ion trap instruments (LTQ-FT/Orbitrap) to characterize and improve linearity and dynamic range of label-free approaches. Quantification based on spectral counts was limited by saturation and ion suppression effects with samples exceeding 100 ng of protein, depending on the instrument setup. In contrast, signal intensities of peptides (peak volumes) selected by a novel correlation-based method (TopCorr-PV) were linear over at least 4 orders of magnitude and allowed for accurate relative quantification of standard proteins spiked into a complex protein background. Application of this procedure to APs of the voltage-gated potassium channel Kv1.1 as a model membrane protein complex unambiguously identified the whole set of known interaction partners together with novel candidates. In addition to discriminating these proteins from background, we could determine efficiency, cross-reactivities, and selection biases of the used purification antibodies. The enhanced dynamic range of the developed quantification procedure appears well suited for sensitive identification of specific protein-protein interactions, detection of antibody-related artifacts, and optimization of AP conditions.


Assuntos
Encéfalo/metabolismo , Cromatografia de Afinidade , Canal de Potássio Kv1.1/análise , Canal de Potássio Kv1.1/isolamento & purificação , Proteômica , Animais , Membrana Celular/metabolismo , Cromatografia Líquida , Análise de Fourier , Canal de Potássio Kv1.1/metabolismo , Camundongos , Ratos , Espectrometria de Massas em Tandem
20.
Cell Rep ; 43(3): 113772, 2024 Mar 26.
Artigo em Inglês | MEDLINE | ID: mdl-38393949

RESUMO

The mitochondrial inner membrane plays central roles in bioenergetics and metabolism and contains several established membrane protein complexes. Here, we report the identification of a mega-complex of the inner membrane, termed mitochondrial multifunctional assembly (MIMAS). Its large size of 3 MDa explains why MIMAS has escaped detection in the analysis of mitochondria so far. MIMAS combines proteins of diverse functions from respiratory chain assembly to metabolite transport, dehydrogenases, and lipid biosynthesis but not the large established supercomplexes of the respiratory chain, ATP synthase, or prohibitin scaffold. MIMAS integrity depends on the non-bilayer phospholipid phosphatidylethanolamine, in contrast to respiratory supercomplexes whose stability depends on cardiolipin. Our findings suggest that MIMAS forms a protein-lipid mega-assembly in the mitochondrial inner membrane that integrates respiratory biogenesis and metabolic processes in a multifunctional platform.


Assuntos
Mitocôndrias , Membranas Mitocondriais , Mitocôndrias/metabolismo , Membranas Mitocondriais/metabolismo , Fosfolipídeos/metabolismo , Transporte de Elétrons , Cardiolipinas/metabolismo
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