RESUMO
Phosphoinositide membrane lipids are ubiquitous low-abundance signaling molecules. They direct many physiological processes that involve ion channels, membrane identification, fusion of membrane vesicles, and vesicular endocytosis. Pools of these lipids are continually broken down and refilled in living cells, and the rates of some of these reactions are strongly accelerated by physiological stimuli. Recent biophysical experiments described here measure and model the kinetics and regulation of these lipid signals in intact cells. Rapid on-line monitoring of phosphoinositide metabolism is made possible by optical tools and electrophysiology. The experiments reviewed here reveal that as for other cellular second messengers, the dynamic turnover and lifetimes of membrane phosphoinositides are measured in seconds, controlling and timing rapid physiological responses, and the signaling is under strong metabolic regulation. The underlying mechanisms of this metabolic regulation remain questions for the future.
Assuntos
Endocitose , Fosfatidilinositóis , Metabolismo dos Lipídeos , Fosfatidilinositóis/metabolismo , Transporte Proteico , Transdução de SinaisRESUMO
This retrospective traces the hypothesis of ion channels from an early statement in a 1970 essay in this journal (Hille, B., 1970, Prog. Biophys. Mol. Biol. 21, 1-32) to its realization today in biophysical, molecular, biochemical, and structural terms. The Na+ and K+ channels of the action potential have been isolated, reconstituted, cloned, mutated, and expressed. They are conformationally flexible, multi-pass glycosylated membrane proteins. Refined atomic structures of several conformational states are known. The discoveries over this half century history illustrate the growth of a field from initial ideas to a mature discipline of biology, physiology, and biomedical science.
Assuntos
Canais Iônicos , Potássio , Canais Iônicos/fisiologia , Íons/metabolismo , Potássio/metabolismo , Estudos Retrospectivos , Sódio/metabolismoRESUMO
Biophysics is a way of approaching biological problems through numbers, physical laws, models, and quantitative logic. In a long scientific career, I have seen the formation and fruition of the ion channel concept through biophysical study. Marvelous discoveries were made as our instruments evolved from vacuum tubes to transistors; computers evolved from the size of an entire building to a few chips inside our instruments; and genome sequencing, gene expression, and atom-level structural biology became accessible to all laboratories. Science is rewarding and exhilarating.
Assuntos
Canais Iônicos , Fenômenos Biofísicos , BiofísicaRESUMO
The pineal gland secretes melatonin principally at night. Regulated by norepinephrine released from sympathetic nerve terminals, adrenergic receptors on pinealocytes activate aralkylamine N-acetyltransferase that converts 5-hydroxytryptamine (5-HT, serotonin) to N-acetylserotonin, the precursor of melatonin. Previous studies from our group and others reveal significant constitutive secretion of 5-HT from pinealocytes. Here, using mass spectrometry, we demonstrated that the 5-HT is secreted primarily via a decynium-22-sensitive equilibrative plasma membrane monoamine transporter instead of by typical exocytotic quantal secretion. Activation of the endogenous 5-HT receptors on pinealocytes evoked an intracellular Ca2+ rise that was blocked by RS-102221, an antagonist of 5-HT2C receptors. Applied 5-HT did not evoke melatonin secretion by itself, but it did potentiate melatonin secretion evoked by submaximal norepinephrine. In addition, RS-102221 reduced the norepinephrine-induced melatonin secretion in strips of pineal gland, even when no exogenous 5-HT was added, suggesting that the 5-HT that is constitutively released from pinealocytes accumulates enough in the tissue to act as an autocrine feedback signal sensitizing melatonin release.
Assuntos
Melatonina/biossíntese , Neurotransmissores/fisiologia , Glândula Pineal/metabolismo , Serotonina/fisiologia , Animais , Exocitose , Proteínas de Ligação ao GTP/metabolismo , Ativação do Canal Iônico , Ligantes , Masculino , Glândula Pineal/citologia , Ratos , Ratos Sprague-Dawley , Transdução de Sinais , Regulação para CimaRESUMO
ß-arrestins regulate many cellular functions including intracellular signaling and desensitization of G protein-coupled receptors (GPCRs). Previous studies show that ß-arrestin signaling and receptor endocytosis are modulated by the plasma membrane phosphoinositide lipid phosphatidylinositol-(4, 5)-bisphosphate (PI(4,5)P2). We found that ß-arrestin also helped promote synthesis of PI(4,5)P2 and up-regulated GPCR endocytosis. We studied these questions with the Gq-coupled protease-activated receptor 2 (PAR2), which activates phospholipase C, desensitizes quickly, and undergoes extensive endocytosis. Phosphoinositides were monitored and controlled in live cells using lipid-specific fluorescent probes and genetic tools. Applying PAR2 agonist initiated depletion of PI(4,5)P2, which then recovered during rapid receptor desensitization, giving way to endocytosis. This endocytosis could be reduced by various manipulations that depleted phosphoinositides again right after phosphoinositide recovery: PI(4)P, a precusor of PI(4,5)P2, could be depleted at either the Golgi or the plasma membrane (PM) using a recruitable lipid 4-phosphatase enzyme and PI(4,5)P2 could be depleted at the PM using a recruitable 5-phosphatase. Endocytosis required the phosphoinositides. Knock-down of ß-arrestin revealed that endogenous ß-arrestin normally doubles the rate of PIP5-kinase (PIP5K) after PAR2 desensitization, boosting PI(4,5)P2-dependent formation of clathrin-coated pits (CCPs) at the PM. Desensitized PAR2 receptors were swiftly immobilized when they encountered CCPs, showing a dwell time of â¼90 s, 100 times longer than for unactivated receptors. PAR2/ß-arrestin complexes eventually accumulated around the edges or across the surface of CCPs promoting transient binding of PIP5K-Iγ. Taken together, ß-arrestins can coordinate potentiation of PIP5K activity at CCPs to induce local PI(4,5)P2 generation that promotes recruitment of PI(4,5)P2-dependent endocytic machinery.
Assuntos
Fosfatidilinositol 4,5-Difosfato/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , beta-Arrestinas/metabolismo , Arrestinas/metabolismo , Membrana Celular/metabolismo , Clatrina/metabolismo , Endocitose/fisiologia , Células HEK293 , Humanos , Fosfatidilinositol 4,5-Difosfato/fisiologia , Fosfatidilinositóis/metabolismo , Fosforilação , Ligação Proteica , Receptor PAR-2/metabolismo , Receptores Acoplados a Proteínas G/fisiologia , Transdução de Sinais , beta-Arrestina 1/metabolismo , beta-Arrestinas/fisiologiaRESUMO
Possible segregation of plasma membrane (PM) phosphoinositide metabolism in membrane lipid domains is not fully understood. We exploited two differently lipidated peptide sequences, L10 and S15, to mark liquid-ordered, cholesterol-rich (Lo) and liquid-disordered, cholesterol-poor (Ld) domains of the PM, often called raft and nonraft domains, respectively. Imaging of the fluorescent labels verified that L10 segregated into cholesterol-rich Lo phases of cooled giant plasma-membrane vesicles (GPMVs), whereas S15 and the dye FAST DiI cosegregated into cholesterol-poor Ld phases. The fluorescent protein markers were used as Förster resonance energy transfer (FRET) pairs in intact cells. An increase of homologous FRET between L10 probes showed that depleting membrane cholesterol shrank Lo domains and enlarged Ld domains, whereas a decrease of L10 FRET showed that adding more cholesterol enlarged Lo and shrank Ld Heterologous FRET signals between the lipid domain probes and phosphoinositide marker proteins suggested that phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] and phosphatidylinositol 4-phosphate (PtdIns4P) are present in both Lo and Ld domains. In kinetic analysis, muscarinic-receptor-activated phospholipase C (PLC) depleted PtdIns(4,5)P2 and PtdIns4P more rapidly and produced diacylglycerol (DAG) more rapidly in Lo than in Ld Further, PtdIns(4,5)P2 was restored more rapidly in Lo than in Ld Thus destruction and restoration of PtdIns(4,5)P2 are faster in Lo than in Ld This suggests that Lo is enriched with both the receptor G protein/PLC pathway and the PtdIns/PI4-kinase/PtdIns4P pathway. The significant kinetic differences of lipid depletion and restoration also mean that exchange of lipids between these domains is much slower than free diffusion predicts.
Assuntos
Microdomínios da Membrana/metabolismo , Peptídeos/metabolismo , Fosfatidilinositol 4,5-Difosfato/metabolismo , Fosfatos de Fosfatidilinositol/metabolismo , Processamento de Proteína Pós-Traducional , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Linhagem Celular Transformada , Colesterol/metabolismo , Difusão , Diglicerídeos/metabolismo , Transferência Ressonante de Energia de Fluorescência , Expressão Gênica , Genes Reporter , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Células HEK293 , Humanos , Cinética , Lipoilação , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Lipídeos de Membrana/metabolismo , Peptídeos/genética , Receptores Muscarínicos/genética , Receptores Muscarínicos/metabolismo , Fosfolipases Tipo C/genética , Fosfolipases Tipo C/metabolismo , Lipossomas Unilamelares/metabolismoRESUMO
The dynamic metabolism of membrane phosphoinositide lipids involves several cellular compartments including the ER, Golgi, and plasma membrane. There are cycles of phosphorylation and dephosphorylation and of synthesis, transfer, and breakdown. The simplified phosphoinositide cycle comprises synthesis of phosphatidylinositol in the ER, transport, and phosphorylation in the Golgi and plasma membranes to generate phosphatidylinositol 4,5-bisphosphate, followed by receptor-stimulated hydrolysis in the plasma membrane and return of the components to the ER for reassembly. Using probes for specific lipid species, we have followed and analyzed the kinetics of several of these events during stimulation of M1 muscarinic receptors coupled to the G-protein Gq. We show that during long continued agonist action, polyphosphorylated inositol lipids are initially depleted but then regenerate while agonist is still present. Experiments and kinetic modeling reveal that the regeneration results from gradual but massive up-regulation of PI 4-kinase pathways rather than from desensitization of receptors. Golgi pools of phosphatidylinositol 4-phosphate and the lipid kinase PI4KIIIα (PI4KA) contribute to this homeostatic regeneration. This powerful acceleration, which may be at the level of enzyme activity or of precursor and product delivery, reveals strong regulatory controls in the phosphoinositide cycle.
Assuntos
1-Fosfatidilinositol 4-Quinase , Membrana Celular/química , Fosfatidilinositol 4,5-Difosfato , Fosfolipases Tipo CRESUMO
Transmembrane 16A (TMEM16A, anoctamin1), 1 of 10 TMEM16 family proteins, is a Cl- channel activated by intracellular Ca2+ and membrane voltage. This channel is also regulated by the membrane phospholipid phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2]. We find that two splice variants of TMEM16A show different sensitivity to endogenous PI(4,5)P2 degradation, where TMEM16A(ac) displays higher channel activity and more current inhibition by PI(4,5)P2 depletion than TMEM16A(a). These two channel isoforms differ in the alternative splicing of the c-segment (exon 13). The current amplitude and PI(4,5)P2 sensitivity of both TMEM16A(ac) and (a) are significantly strengthened by decreased free cytosolic ATP and by conditions that decrease phosphorylation by Ca2+/calmodulin-dependent protein kinase II (CaMKII). Noise analysis suggests that the augmentation of currents is due to a rise of single-channel current (i), but not of channel number (N) or open probability (PO). Mutagenesis points to arginine 486 in the first intracellular loop as a putative binding site for PI(4,5)P2, and to serine 673 in the third intracellular loop as a site for regulatory channel phosphorylation that modulates the action of PI(4,5)P2 In silico simulation suggests how phosphorylation of S673 allosterically and differently changes the structure of the distant PI(4,5)P2-binding site between channel splice variants with and without the c-segment exon. In sum, our study reveals the following: differential regulation of alternatively spliced TMEM16A(ac) and (a) by plasma membrane PI(4,5)P2, modification of these effects by channel phosphorylation, identification of the molecular sites, and mechanistic explanation by in silico simulation.
Assuntos
Processamento Alternativo , Anoctamina-1/genética , Anoctamina-1/metabolismo , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Fosfatidilinositóis/metabolismo , Regulação Alostérica , Animais , Anoctamina-1/química , Sítios de Ligação , Membrana Celular/metabolismo , Regulação da Expressão Gênica , Células HEK293 , Humanos , Ativação do Canal Iônico/efeitos dos fármacos , Camundongos , Modelos Moleculares , Conformação Molecular , Mutagênese Sítio-Dirigida , Fosforilação , Ligação Proteica , Isoformas de Proteínas , Relação Estrutura-AtividadeRESUMO
INTRODUCTION: At night, the pineal gland produces the indoleamines, melatonin, N-acetylserotonin (NAS), and N-acetyltryptamine (NAT). Melatonin is accepted as a hormone of night. Could NAS and NAT serve that role too? METHODS: Concentration-response measurements with overexpressed human melatonin receptors MT1 and MT2 ; mass spectrometry analysis of norepinephrine-stimulated secretions from isolated rat pineal glands; analysis of 24-hour periodic samples of rat blood. RESULTS: We show that NAT and NAS do activate melatonin receptors MT1 and MT2 , although with lower potency than melatonin, and that in vitro, melatonin and NAS are secreted from stimulated, isolated pineal glands in roughly equimolar amounts, but secretion of NAT was much less. All three were found at roughly equal concentrations in blood during the night. However, during the day, serum melatonin fell to very low values creating a high-amplitude circadian rhythm that was absent after pinealectomy, whereas NAS and NAT showed only small or no circadian variation. CONCLUSION: Blood levels of NAS and NAT were insufficient to activate peripheral melatonin receptors, and they were invariant, so they could not serve as circulating hormones of night. However, they could instead act in paracrine circadian fashion near the pineal gland or via other higher-affinity receptors.
Assuntos
Ritmo Circadiano , Glândula Pineal/metabolismo , Receptor MT1 de Melatonina/metabolismo , Receptor MT2 de Melatonina/metabolismo , Serotonina/análogos & derivados , Triptaminas/metabolismo , Animais , Células HEK293 , Humanos , Masculino , Melatonina/metabolismo , Ratos , Ratos Sprague-Dawley , Serotonina/metabolismoRESUMO
Phosphoinositide lipids regulate many cellular processes and are synthesized by lipid kinases. Type I phosphatidylinositol phosphate 5-kinases (PIP5KIs) generate phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2]. Several phosphoinositide-sensitive readouts revealed the nonequivalence of overexpressing PIP5KIß, PIP5KIγ or Ras association domain family 4 (RASSF4), believed to activate PIP5KIs. Mass spectrometry showed that each of these three proteins increased total cellular phosphatidylinositol bisphosphates (PtdInsP2) and trisphosphates (PtdInsP3) at the expense of phosphatidylinositol phosphate (PtdInsP) without changing lipid acyl chains. Analysis of KCNQ2/3 channels and PH domains confirmed an increase in plasma membrane PtdIns(4,5)P2 in response to PIP5KIß or PIP5KIγ overexpression, but RASSF4 required coexpression with PIP5KIγ to increase plasma membrane PtdIns(4,5)P2 Effects on the several steps of store-operated calcium entry (SOCE) were not explained by plasma membrane phosphoinositide increases alone. PIP5KIß and RASSF4 increased STIM1 proximity to the plasma membrane, accelerated STIM1 mobilization and speeded onset of SOCE; however, PIP5KIγ reduced STIM1 recruitment but did not change induced Ca2+ entry. These differences imply actions through different segregated pools of phosphoinositides and specific protein-protein interactions and targeting.This article has an associated First Person interview with the first author of the paper.
Assuntos
Membrana Celular/metabolismo , Fosfatos de Fosfatidilinositol/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Humanos , TransfecçãoRESUMO
Voltage-sensing phosphatases (VSPs) cleave both 3- and 5-phosphates from inositol phospholipids in response to membrane depolarization. When low concentrations of Ciona intestinalis VSP are expressed in Xenopus laevis oocytes, the 5-phosphatase reaction can be observed during large membrane depolarizations. When higher concentrations are expressed, the 5-phosphatase activity is observed with smaller depolarizations, and the 3-phosphatase activity is revealed with strong depolarization. Here we ask whether this apparent induction of 3-phosphatase activity is attributable to the dimerization that has been reported when VSP is expressed at higher concentrations. Using a simple kinetic model, we show that these enzymatic phenomena can be understood as an emergent property of a voltage-dependent enzyme with invariant substrate selectivity operating in the context of endogenous lipid-metabolizing enzymes present in oocytes. Thus, a switch of substrate specificity with dimerization need not be invoked to explain the appearance of 3-phosphatase activity at high VSP concentrations.
Assuntos
Monoéster Fosfórico Hidrolases/metabolismo , Multimerização Proteica , Animais , Humanos , Potenciais da Membrana , Monoéster Fosfórico Hidrolases/química , Especificidade por Substrato , XenopusRESUMO
Polyphosphoinositides (PPIs) are essential phospholipids located in the cytoplasmic leaflet of eukaryotic cell membranes. Despite contributing only a small fraction to the bulk of cellular phospholipids, they make remarkable contributions to practically all aspects of a cell's life and death. They do so by recruiting cytoplasmic proteins/effectors or by interacting with cytoplasmic domains of membrane proteins at the membrane-cytoplasm interface to organize and mold organelle identity. The present study summarizes aspects of our current understanding concerning the metabolism, manipulation, measurement, and intimate roles these lipids play in regulating membrane homeostasis and vital cell signaling reactions in health and disease.
Assuntos
Membrana Celular/metabolismo , Proteínas de Membrana/metabolismo , Fosfatidilinositóis/metabolismo , Transdução de Sinais , Animais , HumanosRESUMO
G protein-coupled seven-transmembrane receptors (GPCRs) mediate responses to hormones, metabolites, lipids, and neurotransmitters at the cell membrane, and so they are prominent drug targets. Although many structural, biochemical, cell biological, and biophysical studies made remarkable progress to understand mechanisms of GPCR signaling, there still are many unanswered questions about arrestin-dependent GPCR signaling. In this chapter, we focus on optical assays to see muscarinic receptor-arrestin interactions with ensemble FRET and single-molecule TIRF imaging in live cells and finally to integrate the information to simulate hypothesized steps in Virtual Cell.
Assuntos
Arrestinas/metabolismo , Transferência Ressonante de Energia de Fluorescência/métodos , Receptores Muscarínicos/metabolismo , Técnicas Biossensoriais , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Células HEK293 , Humanos , Cinética , Ligação ProteicaRESUMO
ß subunits of high voltage-gated Ca2+ (CaV) channels promote cell-surface expression of pore-forming α1 subunits and regulate channel gating through binding to the α-interaction domain (AID) in the first intracellular loop. We addressed the stability of CaV α1B-ß interactions by rapamycin-translocatable CaV ß subunits that allow drug-induced sequestration and uncoupling of the ß subunit from CaV2.2 channel complexes in intact cells. Without CaV α1B/α2δ1, all modified ß subunits, except membrane-tethered ß2a and ß2e, are in the cytosol and rapidly translocate upon rapamycin addition to anchors on target organelles: plasma membrane, mitochondria, or endoplasmic reticulum. In cells coexpressing CaV α1B/α2δ1 subunits, the translocatable ß subunits colocalize at the plasma membrane with α1B and stay there after rapamycin application, indicating that interactions between α1B and bound ß subunits are very stable. However, the interaction becomes dynamic when other competing ß isoforms are coexpressed. Addition of rapamycin, then, switches channel gating and regulation by phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] lipid. Thus, expression of free ß isoforms around the channel reveals a dynamic aspect to the α1B-ß interaction. On the other hand, translocatable ß subunits with AID-binding site mutations are easily dissociated from CaV α1B on the addition of rapamycin, decreasing current amplitude and PI(4,5)P2 sensitivity. Furthermore, the mutations slow CaV2.2 current inactivation and shift the voltage dependence of activation to more positive potentials. Mutated translocatable ß subunits work similarly in CaV2.3 channels. In sum, the strong interaction of CaV α1B-ß subunits can be overcome by other free ß isoforms, permitting dynamic changes in channel properties in intact cells.
Assuntos
Canais de Cálcio Tipo L/metabolismo , Canais de Cálcio Tipo N/metabolismo , Ativação do Canal Iônico/fisiologia , Fosfatidilinositóis/metabolismo , Sirolimo/metabolismo , Animais , Ligação Competitiva , Citosol/metabolismo , Retículo Endoplasmático/metabolismo , Mitocôndrias/metabolismo , Isoformas de Proteínas , Subunidades Proteicas , Transporte Proteico , RatosRESUMO
The stability of organic dyes against photobleaching is critical in single-molecule tracking and localization microscopy. Since oxygen accelerates photobleaching of most organic dyes, glucose oxidase is commonly used to slow dye photobleaching by depleting oxygen. As demonstrated here, pyranose-2-oxidase slows bleaching of Alexa647 dye by â¼20-fold. However, oxygen deprivation may pose severe problems for live cells by reducing mitochondrial oxidative phosphorylation and ATP production. We formulate a method to sustain intracellular ATP levels in the presence of oxygen scavengers. Supplementation with metabolic intermediates including glyceraldehyde, glutamine, and α-ketoisocaproate maintained the intracellular ATP level for at least 10 min by balancing between FADH2 and NADH despite reduced oxygen levels. Furthermore, those metabolites supported ATP-dependent synthesis of phosphatidylinositol 4,5-bisphosphate and internalization of PAR2 receptors. Our method is potentially relevant to other circumstances that involve acute drops of oxygen levels, such as ischemic damage in the brain or heart or tissues for transplantation.
Assuntos
Trifosfato de Adenosina/metabolismo , Oxigênio/metabolismo , Carbocianinas/metabolismo , Linhagem Celular , Flavina-Adenina Dinucleotídeo/análogos & derivados , Flavina-Adenina Dinucleotídeo/metabolismo , Fluorescência , Corantes Fluorescentes/metabolismo , Glucose Oxidase/metabolismo , Glutamina/metabolismo , Células HEK293 , Humanos , Cetoácidos/metabolismo , Microscopia de Fluorescência/métodos , Mitocôndrias/metabolismo , NAD/metabolismo , Fosfatidilinositol 4,5-Difosfato/metabolismo , Fotodegradação , Receptor PAR-2/metabolismoRESUMO
This essay begins with a description of the founding years of Journal of General Physiology (JGP) and a historical overview of the content of the journal. It then turns to key conceptual articles published in JGP that advanced the field of membrane permeation and ion selectivity. Much of this information comes from reading the online archives of JGP and searches in PubMed.
Assuntos
Publicações Periódicas como Assunto/história , Fisiologia/história , Aniversários e Eventos Especiais , História do Século XX , História do Século XXI , Potenciais da MembranaRESUMO
Phosphatidylinositol-4,5-bisphosphate [PI(4,5)P2] is essential for exocytosis. Classical ways of manipulating PI(4,5)P2 levels are slower than its metabolism, making it difficult to distinguish effects of PI(4,5)P2 from those of its metabolites. We developed a membrane-permeant, photoactivatable PI(4,5)P2, which is loaded into cells in an inactive form and activated by light, allowing sub-second increases in PI(4,5)P2 levels. By combining this compound with electrophysiological measurements in mouse adrenal chromaffin cells, we show that PI(4,5)P2 uncaging potentiates exocytosis and identify synaptotagmin-1 (the Ca2+ sensor for exocytosis) and Munc13-2 (a vesicle priming protein) as the relevant effector proteins. PI(4,5)P2 activation of exocytosis did not depend on the PI(4,5)P2-binding CAPS-proteins, suggesting that PI(4,5)P2 uncaging may bypass CAPS-function. Finally, PI(4,5)P2 uncaging triggered the rapid fusion of a subset of readily-releasable vesicles, revealing a rapid role of PI(4,5)P2 in fusion triggering. Thus, optical uncaging of signaling lipids can uncover their rapid effects on cellular processes and identify lipid effectors.
Assuntos
Exocitose , Fosfatidilinositol 4,5-Difosfato/metabolismo , Animais , Proteínas de Transporte/metabolismo , Linhagem Celular , Células Cromafins/metabolismo , Técnicas Citológicas/métodos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas de Membrana/metabolismo , Camundongos , Proteínas do Tecido Nervoso/metabolismo , Sinaptotagmina I/metabolismoRESUMO
CaV-channel dependent activation of BK channels is critical for feedback control of both calcium influx and cell excitability. Here we addressed the functional and spatial interaction between BK and CaV1.3 channels, unique CaV1 channels that activate at low voltages. We found that when BK and CaV1.3 channels were co-expressed in the same cell, BK channels started activating near -50 mV, ~30 mV more negative than for activation of co-expressed BK and high-voltage activated CaV2.2 channels. In addition, single-molecule localization microscopy revealed striking clusters of CaV1.3 channels surrounding clusters of BK channels and forming a multi-channel complex both in a heterologous system and in rat hippocampal and sympathetic neurons. We propose that this spatial arrangement allows tight tracking between local BK channel activation and the gating of CaV1.3 channels at quite negative membrane potentials, facilitating the regulation of neuronal excitability at voltages close to the threshold to fire action potentials.
Assuntos
Canais de Cálcio/metabolismo , Canais de Potássio Ativados por Cálcio de Condutância Alta/metabolismo , Neurônios/química , Neurônios/fisiologia , Animais , Células Cultivadas , Técnicas de Patch-Clamp , RatosRESUMO
Binding of agonists to G-protein-coupled receptors (GPCRs) activates heterotrimeric G proteins and downstream signaling. Agonist-bound GPCRs are then phosphorylated by protein kinases and bound by arrestin to trigger desensitization and endocytosis. Arrestin plays another important signaling function. It recruits and regulates activity of an extracellular signal-regulated kinase (ERK) cascade. However, molecular details and timing of ERK activation remain fundamental unanswered questions that limit understanding of how arrestin-dependent GPCR signaling controls cell functions. Here we validate and model a system that tracks the dynamics of interactions of arrestin with receptors and of ERK activation using optical reporters. Our intermolecular FRET measurements in living cells are consistent with ß-arrestin binding to M1 muscarinic acetylcholine receptors (M1Rs) in two different binding modes, transient and stable. The stable mode persists for minutes after agonist removal. The choice of mode is governed by phosphorylation on key residues in the third intracellular loop of the receptor. We detect a similar intramolecular conformational change in arrestin in either binding mode. It develops within seconds of arrestin binding to the M1 receptor, and it reverses within seconds of arrestin unbinding from the transient binding mode. Furthermore, we observed that, when stably bound to phosphorylated M1R, ß-arrestin scaffolds and activates MEK-dependent ERK. In contrast, when transiently bound, ß-arrestin reduces ERK activity via recruitment of a protein phosphatase. All this ERK signaling develops at the plasma membrane. In this scaffolding hypothesis, a shifting balance between the two arrestin binding modes determines the degree of ERK activation at the membrane.