Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 18 de 18
Filtrar
1.
Cell ; 152(4): 806-17, 2013 Feb 14.
Artículo en Inglés | MEDLINE | ID: mdl-23415228

RESUMEN

Both poikilotherms and homeotherms live longer at lower body temperatures, highlighting a general role of temperature reduction in lifespan extension. However, the underlying mechanisms remain unclear. One prominent model is that cold temperatures reduce the rate of chemical reactions, thereby slowing the rate of aging. This view suggests that cold-dependent lifespan extension is simply a passive thermodynamic process. Here, we challenge this view in C. elegans by showing that genetic programs actively promote longevity at cold temperatures. We find that TRPA-1, a cold-sensitive TRP channel, detects temperature drop in the environment to extend lifespan. This effect requires cold-induced, TRPA-1-mediated calcium influx and a calcium-sensitive PKC that signals to the transcription factor DAF-16/FOXO. Human TRPA1 can functionally substitute for worm TRPA-1 in promoting longevity. Our results reveal a previously unrecognized function for TRP channels, link calcium signaling to longevity, and, importantly, demonstrate that genetic programs contribute to lifespan extension at cold temperatures.


Asunto(s)
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Canales de Calcio/metabolismo , Longevidad , Proteínas del Tejido Nervioso/metabolismo , Sensación Térmica , Canales de Potencial de Receptor Transitorio/metabolismo , Envejecimiento , Animales , Animales Modificados Genéticamente , Canales de Calcio/genética , Señalización del Calcio , Frío , Factores de Transcripción Forkhead , Humanos , Mucosa Intestinal/metabolismo , Proteínas del Tejido Nervioso/genética , Proteína Quinasa C/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Canal Catiónico TRPA1 , Factores de Transcripción/metabolismo , Canales de Potencial de Receptor Transitorio/genética
2.
PLoS Genet ; 14(3): e1007263, 2018 03.
Artículo en Inglés | MEDLINE | ID: mdl-29529030

RESUMEN

The C. elegans ortholog of mammalian calsyntenins, CASY-1, is an evolutionarily conserved type-I transmembrane protein that is highly enriched in the nervous system. Mammalian calsyntenins are strongly expressed at inhibitory synapses, but their role in synapse development and function is still elusive. Here, we report a crucial role for CASY-1 in regulating GABAergic synaptic transmission at the C. elegans neuromuscular junction (NMJ). The shorter isoforms of CASY-1; CASY-1B and CASY-1C, express and function in GABA motor neurons where they regulate GABA neurotransmission. Using pharmacological, behavioral, electrophysiological, optogenetic and imaging approaches we establish that GABA release is compromised at the NMJ in casy-1 mutants. Further, we demonstrate that CASY-1 is required to modulate the transport of GABAergic synaptic vesicle (SV) precursors through a possible interaction with the SV motor protein, UNC-104/KIF1A. This study proposes a possible evolutionarily conserved model for the regulation of GABA synaptic functioning by calsyntenins.


Asunto(s)
Proteínas de Caenorhabditis elegans/fisiología , Caenorhabditis elegans/metabolismo , Neuronas GABAérgicas/metabolismo , Unión Neuromuscular/metabolismo , Isoformas de Proteínas/fisiología , Transmisión Sináptica/fisiología , Ácido gamma-Aminobutírico/metabolismo , Animales , Proteínas de Caenorhabditis elegans/química , Neuronas Motoras/fisiología , Isoformas de Proteínas/química , Transporte de Proteínas
3.
Dev Cell ; 57(6): 750-766.e5, 2022 03 28.
Artículo en Inglés | MEDLINE | ID: mdl-35303431

RESUMEN

Curvature-sensing mechanisms assist proteins in executing particular actions on various membrane organelles. Here, we investigate the functional specificity of curvature-sensing amphipathic motifs in Caenorhabditis elegans through the study of endophilin, an endocytic protein for synaptic vesicle recycling. We generate chimeric endophilin proteins by replacing the endophilin amphipathic motif H0 with other curvature-sensing amphipathic motifs. We find that the role of amphipathic motifs cannot simply be extrapolated from the identity of their parental proteins. For example, the amphipathic motif of the nuclear pore complex protein NUP133 functionally replaces the synaptic role of endophilin H0. Interestingly, non-functional endophilin chimeras have similar defects-producing fewer synaptic vesicles but more endosomes-and this indicates that the curvature-sensing motifs in these chimeras have a common deficiency for reforming synaptic vesicles. Finally, we convert non-functional endophilin chimeras into functional proteins by changing the cationic property of amphipathic motifs, successfully reprogramming the functional specificity of curvature-sensing motifs in vivo.


Asunto(s)
Vesículas Sinápticas , Aciltransferasas/química , Aciltransferasas/fisiología , Secuencias de Aminoácidos , Animales , Caenorhabditis elegans/genética , Proteínas de Complejo Poro Nuclear/metabolismo , Electricidad Estática , Vesículas Sinápticas/metabolismo
4.
Neuron ; 110(5): 824-840.e10, 2022 03 02.
Artículo en Inglés | MEDLINE | ID: mdl-35065714

RESUMEN

Autophagy is a cellular degradation pathway essential for neuronal health and function. Autophagosome biogenesis occurs at synapses, is locally regulated, and increases in response to neuronal activity. The mechanisms that couple autophagosome biogenesis to synaptic activity remain unknown. In this study, we determine that trafficking of ATG-9, the only transmembrane protein in the core autophagy pathway, links the synaptic vesicle cycle with autophagy. ATG-9-positive vesicles in C. elegans are generated from the trans-Golgi network via AP-3-dependent budding and delivered to presynaptic sites. At presynaptic sites, ATG-9 undergoes exo-endocytosis in an activity-dependent manner. Mutations that disrupt endocytosis, including a lesion in synaptojanin 1 associated with Parkinson's disease, result in abnormal ATG-9 accumulation at clathrin-rich synaptic foci and defects in activity-induced presynaptic autophagy. Our findings uncover regulated key steps of ATG-9 trafficking at presynaptic sites and provide evidence that ATG-9 exo-endocytosis couples autophagosome biogenesis at presynaptic sites with the activity-dependent synaptic vesicle cycle.


Asunto(s)
Caenorhabditis elegans , Vesículas Sinápticas , Animales , Autofagia/fisiología , Proteínas Relacionadas con la Autofagia/metabolismo , Caenorhabditis elegans/metabolismo , Endocitosis/fisiología , Terminales Presinápticos/metabolismo , Vesículas Sinápticas/metabolismo
5.
Neuron ; 56(4): 657-69, 2007 Nov 21.
Artículo en Inglés | MEDLINE | ID: mdl-18031683

RESUMEN

The nematode C. elegans provides a powerful model system for exploring the molecular basis of synaptogenesis and neurotransmission. However, the lack of direct functional assays of release processes has largely prevented an in depth understanding of the mechanism of vesicular exocytosis and endocytosis in C. elegans. We address this technical limitation by developing direct electrophysiological assays, including membrane capacitance and amperometry measurements, in primary cultured C. elegans neurons. In addition, we have succeeded in monitoring the docking and fusion of single dense core vesicles (DCVs) employing total internal reflection fluorescence microscopy. With these approaches and mutant perturbation analysis, we provide direct evidence that UNC-31 is required for the docking of DCVs at the plasma membrane. Interestingly, the defect in DCV docking caused by UNC-31 mutation can be fully rescued by PKA activation. We also demonstrate that UNC-31 is required for UNC-13-mediated augmentation of DCV exocytosis.


Asunto(s)
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/metabolismo , Proteínas de Unión al Calcio/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Sistema Nervioso/metabolismo , Neuronas/metabolismo , Vesículas Secretoras/metabolismo , Animales , Caenorhabditis elegans/ultraestructura , Proteínas Portadoras , Membrana Celular/metabolismo , Células Cultivadas , Activación Enzimática/fisiología , Exocitosis/fisiología , Proteínas Fluorescentes Verdes , Membranas Intracelulares/metabolismo , Fusión de Membrana/fisiología , Sistema Nervioso/ultraestructura , Neuronas/ultraestructura , Neurosecreción/fisiología , Neurotransmisores/metabolismo , Vesículas Secretoras/ultraestructura , Serotonina/metabolismo , Membranas Sinápticas/metabolismo , Transmisión Sináptica/fisiología
6.
Biochem Biophys Res Commun ; 371(4): 781-6, 2008 Jul 11.
Artículo en Inglés | MEDLINE | ID: mdl-18468511

RESUMEN

Synaptotagmins (Syts) are calcium-binding proteins which are conserved from nematodes to humans. Fifteen Syts have been identified in mammalian species. Syt I is recognized as a Ca(2+) sensor for the synchronized release of synaptic vesicles in some types of neurons, but its role in the secretion of dense core vesicles (DCVs) remains unclear. The function of Syt IV is of particular interest because it is rapidly up-regulated by chronic depolarization and seizures. Using RNAi-mediated gene silencing, we have explored the role of Syt I and IV on secretion in a pituitary gonadotrope cell line. Downregulation of Syt IV clearly reduced Ca(2+)-triggered exocytosis of dense core vesicles (DCVs) in LbetaT2 cells. Syt I silencing, however, had no effect on vesicular release.


Asunto(s)
Exocitosis , Vesículas Secretoras/metabolismo , Sinaptotagminas/fisiología , Animales , Calcio/farmacología , Línea Celular , Regulación hacia Abajo , Exocitosis/efectos de los fármacos , Exocitosis/genética , Ratones , Interferencia de ARN , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/farmacología , Vesículas Secretoras/efectos de los fármacos , Sinaptotagmina I/genética , Sinaptotagmina I/metabolismo , Sinaptotagmina I/fisiología , Sinaptotagminas/genética , Sinaptotagminas/metabolismo
7.
Neuron ; 100(1): 150-166.e4, 2018 10 10.
Artículo en Inglés | MEDLINE | ID: mdl-30269993

RESUMEN

Synapse formation defines neuronal connectivity and is thus essential for neuronal circuit assembly. Trans-synaptic interactions of cell adhesion molecules are thought to induce synapse assembly. Here we demonstrate that a recently discovered and conserved short form of neurexin, γ-neurexin, which lacks canonical extracellular domains, is nonetheless sufficient to promote presynaptic assembly in the nematode C. elegans. γ- but not α-neurexin is required for assembling active zone components, recruiting synaptic vesicles, and clustering calcium channels at release sites to promote evoked synaptic transmission. Furthermore, we find that neurexin functions in parallel with the transmembrane receptor Frizzled, as the absence of both proteins leads to an enhanced phenotype-the loss of most synapses. Frizzled's pro-synaptogenic function is independent of its ligand, Wnt. Wnt binding instead eliminates synapses by inducing Frizzled's endocytosis and the downregulation of neurexin. These results reveal how pro- and anti-synaptogenic factors converge to precisely sculpt circuit formation in vivo.


Asunto(s)
Proteínas de Caenorhabditis elegans/metabolismo , Moléculas de Adhesión Celular Neuronal/metabolismo , Receptores Frizzled/metabolismo , Neurogénesis/fisiología , Sinapsis/metabolismo , Transmisión Sináptica/fisiología , Animales , Caenorhabditis elegans , Endocitosis/fisiología , Neuronas Motoras/metabolismo , Isoformas de Proteínas
8.
Elife ; 62017 03 28.
Artículo en Inglés | MEDLINE | ID: mdl-28349862

RESUMEN

Animals with complex brains can discriminate the spatial arrangement of physical features in the environment. It is unknown whether such sensitivity to spatial patterns can be accomplished in simpler nervous systems that lack long-range sensory modalities such as vision and hearing. Here we show that the nematode Caenorhabditis elegans can discriminate spatial patterns in its surroundings, despite having a nervous system of only 302 neurons. This spatial pattern selectivity requires touch-dependent dopamine signaling, including the mechanosensory TRP-4 channel in dopaminergic neurons and the D2-like dopamine receptor DOP-3. We find that spatial pattern selectivity varies significantly among C. elegans wild isolates. Electrophysiological recordings show that natural variations in TRP-4 reduce the mechanosensitivity of dopaminergic neurons. Polymorphic substitutions in either TRP-4 or DOP-3 alter the selectivity of spatial patterns. Together, these results demonstrate an ancestral role for dopamine signaling in tuning spatial pattern preferences in a simple nervous system.


Asunto(s)
Conducta Animal , Caenorhabditis elegans/fisiología , Neuronas Dopaminérgicas/fisiología , Orientación Espacial , Animales
9.
Mol Biol Cell ; 27(13): 2119-32, 2016 07 01.
Artículo en Inglés | MEDLINE | ID: mdl-27170174

RESUMEN

Numerous proteins act in concert to sculpt membrane compartments for cell signaling and metabolism. These proteins may act as curvature sensors, membrane benders, and scaffolding molecules. Here we show that endophilin, a critical protein for rapid endocytosis, quickly transforms from a curvature sensor into an active bender upon membrane association. We find that local membrane deformation does not occur until endophilin inserts its amphipathic helices into lipid bilayers, supporting an active bending mechanism through wedging. Our time-course studies show that endophilin continues to drive membrane changes on a seconds-to-minutes time scale, indicating that the duration of endocytosis events constrains the mode of endophilin action. Finally, we find a requirement of coordinated activities between wedging and scaffolding for endophilin to produce stable membrane tubules in vitro and to promote synaptic activity in vivo. Together these data demonstrate that endophilin is a multifaceted molecule that precisely integrates activities of sensing, bending, and stabilizing curvature to sculpt membranes with speed.


Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas Adaptadoras Transductoras de Señales/fisiología , Animales , Membrana Celular/metabolismo , Endocitosis/genética , Endocitosis/fisiología , Membrana Dobles de Lípidos/metabolismo , Membranas/metabolismo , Ratones , Modelos Moleculares , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Factores de Tiempo
10.
J Gen Physiol ; 124(6): 653-62, 2004 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-15572345

RESUMEN

Glucose and other secretagogues are thought to activate a variety of protein kinases. This study was designed to unravel the sites of action of protein kinase A (PKA) and protein kinase C (PKC) in modulating insulin secretion. By using high time resolution measurements of membrane capacitance and flash photolysis of caged Ca(2+), we characterize three kinetically different pools of vesicles in rat pancreatic beta-cells, namely, a highly calcium-sensitive pool (HCSP), a readily releasable pool (RRP), and a reserve pool. The size of the HCSP is approximately 20 fF under resting conditions, but is dramatically increased by application of either phorbol esters or forskolin. Phorbol esters and forskolin also increase the size of RRP to a lesser extent. The augmenting effect of phorbol esters or forskolin is blocked by various PKC or PKA inhibitors, indicating the involvement of these kinases. The effects of PKC and PKA on the size of the HCSP are not additive, suggesting a convergent mechanism. Using a protocol where membrane depolarization is combined with photorelease of Ca(2+), we find that the HCSP is a distinct population of vesicles from those colocalized with Ca(2+) channels. We propose that PKA and PKC promote insulin secretion by increasing the number of vesicles that are highly sensitive to Ca(2+).


Asunto(s)
Canales de Calcio/fisiología , Calcio/metabolismo , Exocitosis/fisiología , Insulina/metabolismo , Islotes Pancreáticos/fisiología , Proteínas Quinasas/metabolismo , Vesículas Transportadoras/metabolismo , Animales , Señalización del Calcio/fisiología , Células Cultivadas , Activación Enzimática , Secreción de Insulina , Masculino , Ratas , Ratas Wistar
11.
Elife ; 42015 Apr 28.
Artículo en Inglés | MEDLINE | ID: mdl-25918845

RESUMEN

Synaptojanin and endophilin represent a classic pair of endocytic proteins that exhibit coordinated action during rapid synaptic vesicle endocytosis. Current models suggest that synaptojanin activity is tightly associated with endophilin through high-affinity binding between the synaptojanin proline-rich domain (PRD) and the endophilin SH3 domain. Surprisingly, we find that truncated synaptojanin lacking the PRD domain sustains normal synaptic transmission, indicating that synaptojanin's core function in vivo resides in the remaining two domains that contain phosphoinositide-phosphatase activities: an N-terminal Sac1 phosphatase domain and a 5-phosphatase domain. We further show that the Sac1 domain plays an unexpected role in targeting synaptojanin to synapses. The requirement for Sac1 is bypassed by tethering the synaptojanin 5-phophatase to the endophilin membrane-bending Bin-Amphiphysin-Rvs (BAR) domain. Together, our results uncover an unexpected role for the Sac1 domain in vivo in supporting coincident action between synaptojanin and endophilin at synapses.


Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/genética , Endocitosis/genética , Proteínas del Tejido Nervioso/genética , Monoéster Fosfórico Hidrolasas/genética , Sinapsis/genética , Transmisión Sináptica/genética , Animales , Animales Modificados Genéticamente , Caenorhabditis elegans , Proteínas de Unión al ADN/genética , Técnicas de Transferencia de Gen , Microinyecciones , Transmisión Sináptica/fisiología , Transposasas/genética
12.
Elife ; 32014 Nov 10.
Artículo en Inglés | MEDLINE | ID: mdl-25383924

RESUMEN

The presynaptic protein complexin (CPX) is a critical regulator of synaptic vesicle fusion, but the mechanisms underlying its regulatory effects are not well understood. Its highly conserved central helix (CH) directly binds the ternary SNARE complex and is required for all known CPX functions. The adjacent accessory helix (AH) is not conserved despite also playing an important role in CPX function, and numerous models for its mechanism have been proposed. We examined the impact of AH mutations and chimeras on CPX function in vivo and in vitro using C. elegans. The mouse AH fully restored function when substituted into worm CPX suggesting its mechanism is evolutionarily conserved. CPX inhibitory function was impaired when helix propagation into the CH was disrupted whereas replacing the AH with a non-native helical sequence restored CPX function. We propose that the AH operates by stabilizing CH secondary structure rather than through protein or lipid interactions.

13.
Neuron ; 77(2): 323-34, 2013 Jan 23.
Artículo en Inglés | MEDLINE | ID: mdl-23352168

RESUMEN

Synapses continually replenish their synaptic vesicle (SV) pools while suppressing spontaneous fusion events, thus maintaining a high dynamic range in response to physiological stimuli. The presynaptic protein complexin can both promote and inhibit fusion through interactions between its α-helical domain and the SNARE complex. In addition, complexin's C-terminal half is required for the inhibition of spontaneous fusion in worm, fly, and mouse, although the molecular mechanism remains unexplained. We show here that complexin's C-terminal domain binds lipids through a novel protein motif, permitting complexin to inhibit spontaneous exocytosis in vivo by targeting complexin to SVs. We propose that the SV pool serves as a platform to sequester and position complexin where it can intercept the rapidly assembling SNAREs and control the rate of spontaneous fusion.


Asunto(s)
Proteínas Adaptadoras del Transporte Vesicular/metabolismo , Fusión de Membrana/genética , Proteínas del Tejido Nervioso/metabolismo , Vesículas Sinápticas/metabolismo , Proteínas Adaptadoras del Transporte Vesicular/genética , Secuencia de Aminoácidos , Animales , Caenorhabditis elegans , Drosophila , Ratones , Datos de Secuencia Molecular , Proteínas del Tejido Nervioso/genética , Unión Proteica/genética , Estructura Terciaria de Proteína/genética , Vesículas Sinápticas/genética
14.
Nat Neurosci ; 13(6): 715-22, 2010 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-20436480

RESUMEN

The eyeless animal C. elegans is able to sense light and engages in phototaxis behavior that is mediated by photoreceptor cells. However, the molecular and cellular mechanisms underlying phototransduction in C. elegans remain largely unclear. By recording the photoreceptor neuron ASJ in wild-type and various mutant worms, we found that phototransduction in ASJ is a G protein-mediated process and requires membrane-associated guanylate cyclases, but not typical phosphodiesterases. In addition, we found that C. elegans phototransduction requires LITE-1, a candidate photoreceptor protein known to be a member of the invertebrate taste receptor family. Our genetic, pharmacological and electrophysiological data suggest a model in which LITE-1 transduces light signals in ASJ via G protein signaling, which leads to upregulation of the second messenger cGMP, followed by opening of cGMP-sensitive CNG channels and stimulation of photoreceptor cells. Our results identify a phototransduction cascade in C. elegans and implicate the function of a 'taste receptor' in phototransduction.


Asunto(s)
Proteínas de Caenorhabditis elegans/metabolismo , GMP Cíclico/metabolismo , Proteínas de Unión al GTP/metabolismo , Fototransducción/fisiología , Proteínas de la Membrana/metabolismo , Células Fotorreceptoras de Invertebrados/fisiología , Animales , Animales Modificados Genéticamente , Caenorhabditis elegans , Proteínas de Caenorhabditis elegans/genética , Canales Catiónicos Regulados por Nucleótidos Cíclicos/metabolismo , Guanilato Ciclasa/metabolismo , Potenciales de la Membrana/fisiología , Proteínas de la Membrana/genética , Modelos Neurológicos , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Neuronas/fisiología , Hidrolasas Diéster Fosfóricas/metabolismo , Estimulación Luminosa , Transducción de Señal , Regulación hacia Arriba
15.
Biochem Biophys Res Commun ; 360(3): 609-14, 2007 Aug 31.
Artículo en Inglés | MEDLINE | ID: mdl-17617378

RESUMEN

Syntaxin1A and Munc18-1 play essential roles in exocytosis. However, the molecular mechanism and the functional roles of their interaction in insulin secretion remain to be explored. Using membrane capacitance measurement, we examine effect of overexpressing Munc18-1 on exocytosis in pancreatic beta cells. The results show that Munc18-1 negatively regulates vesicle fusion. To probe the interaction between Munc18-1 and Syntaxin1A, Munc18-1-Tdimer2 and EGFP-Syntaxin1A were co-transfected into INS-1 cells. FRET measurement confirmed that Munc18-1 interacted with wild type Syntaxin 1A, but not the constitutively open form (DM) of Syntaxin1A. Overexpressing DM in primary pancreatic beta cells augmented insulin secretion, and this effect can overcome the inhibitory effect of Munc18-1 overexpression. We propose that Munc18-1 inhibitis the SNARE complex assembly by stabilizing Syntaxin1A in a closed conformation in vesicle priming process, therefore negatively regulates insulin secretion.


Asunto(s)
Células Secretoras de Insulina/metabolismo , Insulina/metabolismo , Proteínas Munc18/metabolismo , Proteínas Qa-SNARE/metabolismo , Sintaxina 1/metabolismo , Animales , Células Cultivadas , Transferencia Resonante de Energía de Fluorescencia , Secreción de Insulina , Masculino , Proteínas Munc18/genética , Ratas , Proteínas Recombinantes de Fusión/metabolismo
16.
Acta Pharmacol Sin ; 27(7): 933-8, 2006 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-16787579

RESUMEN

AIM: To characterize Ca2+-stimulated exocytosis in isolated mouse pancreatic beta cells. METHODS: An improved method was described for isolation of mouse pancreatic beta cells by collagenase P. The Ca2+ channel current and the membrane capacitance were examined by using the whole-cell patch clamp recording technique. RESULTS: Using depolarization and flash photolysis of caged Ca2+ to induce Ca2+-dependent exocytosis in beta cell from KM mouse, we have explored the characteristics of the Ca2+ channel current and the relationship between Ca2+ signals and exocytosis. The averaged peak Ca2+ current measured at +20 mV was -60+/-6 pA (n=13). CONCLUSION: We characterized three kinetically different pools of vesicles in mouse pancreatic beta cells, namely an immediately releasable pool, a readily releasable pool, and a reserve pool.


Asunto(s)
Canales de Calcio Tipo L/metabolismo , Señalización del Calcio , Calcio/metabolismo , Exocitosis/fisiología , Células Secretoras de Insulina/metabolismo , Animales , Calcio/fisiología , Células Cultivadas , Endocitosis/fisiología , Células Secretoras de Insulina/citología , Masculino , Ratones
17.
Acta Biochim Biophys Sin (Shanghai) ; 38(2): 136-41, 2006 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-16474905

RESUMEN

Adenosine triphosphate (ATP)-sensitive K+ (KATP) channels regulate many cellular functions by coupling the metabolic state of the cell to the changes in membrane potential. Truncation of C-terminal 26 amino acid residues of Kir6.2 protein (Kir6.2DeltaC26) deletes its endoplasmic reticulum retention signal, allowing functional expression of Kir6.2 in the absence of sulfonylurea receptor subunit. pEGFP-Kir6.2DeltaC26 and pKir6.2DeltaC26-IRES2-EGFP expression plasmids were constructed and transfected into HEK293 cells. We identified that Kir6.2DeltaC26 was localized on the plasma membrane and trafficked to the plasmalemma by means of constitutive exocytosis of Kir6.2DeltaC26 transport vesicles, using epi-fluorescence and total internal reflection fluorescence microscopy. Our electrophysiological data showed that Kir6.2DeltaC26 alone expressed KATP currents, whereas EGFP-Kir6.2DeltaC26 fusion protein displayed no KATP channel activity.


Asunto(s)
Adenosina Trifosfato/metabolismo , Membrana Celular/metabolismo , Exocitosis/fisiología , Canales de Potasio de Rectificación Interna/metabolismo , Transportadoras de Casetes de Unión a ATP/metabolismo , Animales , Secuencia de Bases , Transporte Biológico , Células Cultivadas , ADN Complementario/química , ADN Complementario/genética , Electrofisiología , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Microscopía Fluorescente , Plásmidos/genética , Canales de Potasio/metabolismo , Receptores de Droga/metabolismo , Receptores de Sulfonilureas , Factores de Tiempo
SELECCIÓN DE REFERENCIAS
DETALLE DE LA BÚSQUEDA