RESUMEN
1. Fast synaptic transmission is triggered by the activation of presynaptic Ca2+ channels which can be inhibited by Gbetagamma subunits via G protein-coupled receptors (GPCR). Regulators of G protein signalling (RGS) proteins are GTPase-accelerating proteins (GAPs), which are responsible for >100-fold increases in the GTPase activity of G proteins and might be involved in the regulation of presynaptic Ca2+ channels. In this study we investigated the effects of RGS2 on G protein modulation of recombinant P/Q-type channels expressed in a human embryonic kidney (HEK293) cell line using whole-cell recordings. 2. RGS2 markedly accelerates transmitter-mediated inhibition and recovery from inhibition of Ba2+ currents (IBa) through P/Q-type channels heterologously expressed with the muscarinic acetylcholine receptor M2 (mAChR M2). 3. Both RGS2 and RGS4 modulate the prepulse facilitation properties of P/Q-type Ca2+ channels. G protein reinhibition is accelerated, while release from inhibition is slowed. These kinetics depend on the availability of G protein alpha and betagamma subunits which is altered by RGS proteins. 4. RGS proteins unmask the Ca2+ channel beta subunit modulation of Ca2+ channel G protein inhibition. In the presence of RGS2, P/Q-type channels containing the beta2a and beta3 subunits reveal significantly altered kinetics of G protein modulation and increased facilitation compared to Ca2+ channels coexpressed with the beta1b or beta4 subunit.
Asunto(s)
Canales de Calcio Tipo P/metabolismo , Canales de Calcio Tipo Q/metabolismo , Proteínas de Unión al GTP/metabolismo , Riñón/metabolismo , Proteínas RGS/metabolismo , Bario/farmacología , Canales de Calcio/genética , Canales de Calcio/metabolismo , Canales de Calcio Tipo N , Canales de Calcio Tipo P/efectos de los fármacos , Canales de Calcio Tipo P/genética , Canales de Calcio Tipo Q/efectos de los fármacos , Canales de Calcio Tipo Q/genética , Línea Celular , Proteínas de Unión al GTP/farmacología , Guanosina 5'-O-(3-Tiotrifosfato)/farmacología , Humanos , Activación del Canal Iónico/efectos de los fármacos , Riñón/citología , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Técnicas de Placa-Clamp , Fosfoproteínas/biosíntesis , Subunidades de Proteína , Proteínas RGS/farmacología , Tiempo de Reacción/efectos de los fármacos , Receptor Muscarínico M2 , Receptores Muscarínicos/metabolismo , Proteínas Recombinantes/efectos de los fármacos , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Transducción de Señal/efectos de los fármacosRESUMEN
G-protein regulated inward-rectifier potassium channels (GIRK) are part of a superfamily of inward-rectifier K+ channels which includes seven family members. To date four GIRK subunits, designated GIRK1-4 (also designated Kir3.1-4), have been identified in mammals, and GIRK5 has been found in Xenopus oocytes. GIRK channels exist in vivo both as homotetramers and heterotetramers. In contrast to the other mammalian GIRK family members, GIRK1 can not form functional channels by itself and has to assemble with GIRK2, 3 or 4. As the name implies, GIRK channels are modulated by G-proteins; they are also modulated by phosphatidylinositol 4,5-bisphosphate, intracellular sodium, ethanol and mechanical stretch. Recently a family of GTPase activating proteins known as regulators of G-protein signaling were shown to be the missing link for the fast deactivation kinetics of GIRK channels in native cells, which contrast with the slow kinetics observed in heterologously expressed channels. GIRK1, 2 and 3 are highly abundant in brain, while GIRK4 has limited distribution. Here, GIRK1/2 seems to be the predominant heterotetramer. In general, neuronal GIRK channels are involved in the regulation of the excitability of neurons and may contribute to the resting potential. Interestingly, only the GIRK1 and 4 subunits are distributed in the atrial and sinoatrial node cells of the heart and are involved in the regulation of cardiac rate. Our main objective of this review is to assess the current understanding of the G-protein modulation of GIRK channels and their physiological importance in mammals.
Asunto(s)
Proteínas de Unión al GTP/fisiología , Activación del Canal Iónico/fisiología , Canales de Potasio/fisiología , Potasio/metabolismo , Transducción de Señal/fisiología , Animales , Células CHO , Cricetinae , Guanosina Trifosfato/fisiología , Humanos , Activación del Canal Iónico/efectos de los fármacos , Transporte Iónico/efectos de los fármacos , Transporte Iónico/fisiología , Ratones , Ratones Noqueados , Proteínas del Tejido Nervioso/efectos de los fármacos , Proteínas del Tejido Nervioso/fisiología , Neuronas/metabolismo , Fosfatidilinositol 4,5-Difosfato/fisiología , Canales de Potasio/efectos de los fármacos , Proteínas RGS/fisiología , Receptores Muscarínicos/efectos de los fármacos , Receptores Muscarínicos/fisiología , Receptores Purinérgicos/efectos de los fármacos , Receptores Purinérgicos/fisiología , Proteínas Recombinantes de Fusión/fisiología , Transducción de Señal/efectos de los fármacos , Factores de Virulencia de Bordetella/farmacología , XenopusRESUMEN
G protein regulated inward rectifying potassium channels (GIRKs) are activated by G protein coupled receptors (GPCRs) via the G protein betagamma subunits. However, little is known about the effects of different GPCRs on the deactivation kinetics of transmitter-mediated GIRK currents. In the present study we investigated the influence of different GPCRs in the presence and absence of RGS proteins on the deactivation kinetics of GIRK channels by coexpressing the recombinant protein subunits in Xenopus oocytes. The stimulation of both G(i/o)- and G(q)-coupled pathways accelerated GIRK deactivation. GIRK currents deactivated faster upon stimulation of G(i/o)- and G(q)-coupled pathways by P(2)Y(2) receptors (P(2)Y(2)Rs) than upon activation of the G(i/o)-coupled pathway alone via muscarinic acetylcholine receptor M2 (M(2) mAChRs). This acceleration was found to be dependent on phospholipase C (PLC) and protein kinase C (PKC) activities and intracellular calcium. With the assumption that RGS2 has a higher affinity for Galpha(q) than Galpha(i/o), we demonstrated that the deactivation kinetics of GIRK channels can be differentially regulated by the relative amount of RGS proteins. These data indicate that transmitter-mediated deactivation of GIRK currents is modulated by crosstalk between G(i/o)- and G(q)-coupled pathways.
Asunto(s)
Proteínas de Unión al GTP Heterotriméricas/metabolismo , Canales de Potasio de Rectificación Interna , Canales de Potasio/metabolismo , Animales , Calcio/fisiología , Canales de Cloruro/efectos de los fármacos , Canales de Cloruro/metabolismo , Clonación Molecular , Canales de Potasio Rectificados Internamente Asociados a la Proteína G , Subunidades alfa de la Proteína de Unión al GTP Gq-G11 , Humanos , Bloqueadores de los Canales de Potasio , Proteína Quinasa C/metabolismo , Receptor Muscarínico M2 , Receptores Muscarínicos/metabolismo , Receptores Purinérgicos P2/metabolismo , Receptores Purinérgicos P2Y2 , Fosfolipasas de Tipo C/metabolismo , Xenopus laevisRESUMEN
Neurotransmitter release is triggered by the influx of Ca(2+) into the presynaptic terminal through voltage gated Ca(2+)-channels. The shape of the presynaptic Ca(2+) signal largely determines the amount of released quanta and thus the size of the synaptic response. Ca(2+)-channel function is modulated in particular by the auxiliary beta-subunits that interact intracellularly with the pore-forming alpha(1)-subunit. Using retrovirus-mediated gene transfer in cultured hippocampal neurons, we demonstrate that functional GFP-beta(4) constructs colocalize with the synaptic vesicle marker synaptobrevin II and endogenous P/Q-type channels, indicating that beta(4)-subunits are localized to synaptic sites. Costaining with the dendritic marker MAP2 revealed that the beta(4)-subunit is transported to dendrites as well as axons. The nonconserved amino- and carboxyl-termini of the beta(4)-subunit were found to target the protein to the synapse. Physiological measurements in autaptic hippocampal neurons infected with green fluorescent protein (GFP)-beta(4) revealed an increase in both excitatory post-synaptic current amplitude and paired pulse facilitation ratio, whereas the GFP-beta(4) mutant, GFP-beta(4)(Delta50-407), which demonstrated a cytosolic localization pattern, did not alter these synaptic properties. In summary, our data suggest a pre-synaptic function of the Ca(2+)-channel beta(4)-subunit in synaptic transmission.
Asunto(s)
Canales de Calcio/fisiología , Hipocampo/fisiología , Sinapsis/metabolismo , Secuencia de Bases , Canales de Calcio/química , Línea Celular , Cartilla de ADN , Hipocampo/citología , Humanos , Inmunohistoquímica , Potenciales de la Membrana , Sinapsis/fisiologíaRESUMEN
1. The activation of G protein-regulated inward rectifying potassium (GIRK) channels is modulated by G protein-coupled receptors (GPCRs) via the G protein betagamma subunits and is accelerated by regulators of G protein signalling (RGS). In the present study we investigated the ratio dependence of receptor-mediated activation and deactivation and the influence of new members of the RGS protein family on GIRK currents by coexpressing the recombinant protein subunits in Xenopus oocytes and further analysis of the whole cell currents. 2. The activation of GIRK channels by the muscarinic acetylcholine receptor M2 (M2 mAChR) is strongly dependent on the ratio of receptor to channel in Xenopus oocytes. The increase and on-rate of the amplified current is affected by this ratio. An excess of receptor over channel is necessary for current amplification, while the reverse excess of channel over receptor abolishes the effect. 3. The speed of receptor-mediated activation of GIRK currents is accelerated for a high ratio of receptor to channel, while the time of deactivation is independent of this ratio. 4. Coexpression of RGS2, 5 and 8 accelerates the speed for ACh-mediated activation and deactivation of GIRK1/2 and GIRK1/4 currents. Thereby the receptor/channel/RGS ratio determines the amount of current amplification. 5. Bordetella pertussis toxin completely abolished ACh-mediated current amplification of GIRK channels coexpressed with or without RGS2. 6. Two single point mutations in the RGS2 protein (RGS2(N109S) and RGS2(L180F)) reduced the acceleration of current amplification after ACh application on GIRK1/4 channels compared with RGS2 wild-type protein.
Asunto(s)
Proteínas de Unión al GTP/fisiología , Canales de Potasio de Rectificación Interna , Proteínas/fisiología , Proteínas RGS , Proteínas de Xenopus , Secuencia de Aminoácidos/genética , Animales , Conductividad Eléctrica , Femenino , Proteínas Activadoras de GTPasa , Ratones , Datos de Secuencia Molecular , Oocitos/metabolismo , Oocitos/fisiología , Toxina del Pertussis , Mutación Puntual/fisiología , Canales de Potasio/metabolismo , Proteínas/genética , Ratas , Receptores Muscarínicos/fisiología , Proteínas Recombinantes , Factores de Virulencia de Bordetella/farmacología , Xenopus laevis/metabolismoRESUMEN
Axonal growth cones navigate long distances along specific pathways to establish complex patterns of neuronal connections. A growing number of signals have been identified that participate in these steering decisions. This review will concentrate on a large and growing family of chemorepellents, the semaphorins. This family contains both secreted and membrane-bound proteins expressed in many neuronal and non-neuronal tissues of invertebrates and vertebrates. Ongoing studies have given us a better understanding of how their highly conserved signalling system is involved in patterning neuronal connections.
Asunto(s)
Comunicación Celular/fisiología , Proteínas de la Membrana/fisiología , Proteínas del Tejido Nervioso/fisiología , Neuronas/fisiología , Transducción de Señal , Animales , HumanosRESUMEN
Nerve growth factor (NGF) functions as a progression factor with both mitogenic and antimitogenic activities. When PC12 cells are treated with NGF, they advance to the G1 stage of the cell cycle before they differentiate. The correlation between cessation of proliferation and differentiation suggests that the antimitotic activity of NGF may be obligatory for differentiation. Although epidermal growth factor- (EGF) and NGF-treated PC12 cells share several common properties, including activation of the mitogen-activated protein (MAP) kinase pathway and induction of immediate early genes, EGF is mitogenic for PC12 cells and does not normally stimulate differentiation. However, combinations of EGF and low levels of cAMP stimulate differentiation even though neither agent alone does (Mark, M. D., Liu, Y., Wong, S. T., Hinds, T. R., and Storm, D.R. (1995) J. Cell Biol. 130, 701-710). Since EGF is mitogenic for PC12 cells and differentiation may not occur until proliferation is inhibited, differentiation caused by cAMP and EGF may be due to the antiproliferative activity of cAMP. To test this hypothesis, we examined the effect of EGF or combinations of EGF and cAMP on PC12 cell proliferation. EGF alone stimulated proliferation of PC12 cells and increased the levels of several cell cycle progression factors including cdk2, cdk4, and cyclin B1. Cyclic AMP inhibited the EGF-stimulated increases in cell cycle progression factors as well as proliferation. Other antiproliferative agents including rapamycin, mimosine, and nitric oxide agonists also synergized with EGF to stimulate differentiation. These data indicate that the coupling of antiproliferative signals with EGF modifies the biological properties of EGF and converts it to a differentiating growth factor.
Asunto(s)
Quinasas CDC2-CDC28 , AMP Cíclico/metabolismo , Ciclina B , Factor de Crecimiento Epidérmico/farmacología , Neuronas/citología , Proteínas Proto-Oncogénicas , Animales , Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , Diferenciación Celular/efectos de los fármacos , División Celular/efectos de los fármacos , Colforsina/farmacología , Ciclina B1 , Quinasa 2 Dependiente de la Ciclina , Quinasa 4 Dependiente de la Ciclina , Quinasas Ciclina-Dependientes/metabolismo , Ciclinas/metabolismo , Replicación del ADN/efectos de los fármacos , Sinergismo Farmacológico , Fase G1 , Factores de Crecimiento Nervioso/farmacología , Neuronas/efectos de los fármacos , Células PC12 , Proteínas Serina-Treonina Quinasas/metabolismo , RatasRESUMEN
Combinations of epidermal growth factor (EGF) and KCl stimulate differentiation in PC12 cells, independent of extracellular calcium [Mark et al., Stimulation of neurite outgrowth in PC12 cells by EGF and KCl depolarization: a Ca2+-independent phenomenon, J. Cell Biol., 130 (1995) 701-710]. Since EGF is a proliferative agent that normally does not stimulate differentiation of PC12 cells, we hypothesize that KCl plus EGF may cause differentiation because of the anti-proliferative activity of KCl. Here we report that treatment of PC12 cells with KCl plus EGF resulted in a significant decrease in proliferation and DNA synthesis compared with cells treated with EGF alone. In addition, KCl significantly reduced the EGF-induced expression of cell cycle progression factors cdk2, cdk4, cyclin B1 and PCNA. These data suggest that the anti-proliferative activity of KCl may convert EGF from a proliferative factor to a progression factor.
Asunto(s)
Quinasas CDC2-CDC28 , Ciclina B , Factor de Crecimiento Epidérmico/farmacología , Neuritas/fisiología , Cloruro de Potasio/farmacología , Proteínas Proto-Oncogénicas , Animales , Ciclo Celular/efectos de los fármacos , Diferenciación Celular/efectos de los fármacos , División Celular/efectos de los fármacos , Ciclina B1 , Quinasa 2 Dependiente de la Ciclina , Quinasa 4 Dependiente de la Ciclina , Quinasas Ciclina-Dependientes/biosíntesis , Ciclinas/biosíntesis , Replicación del ADN/efectos de los fármacos , Cinética , Neuritas/efectos de los fármacos , Células PC12 , Antígeno Nuclear de Célula en Proliferación/biosíntesis , Proteínas Serina-Treonina Quinasas/biosíntesis , Ratas , Timidina/metabolismoRESUMEN
MAP kinase activity is necessary for growth factor induction of neurite outgrowth in PC12 cells. Although NGF and EGF both stimulate MAP kinase activity, EGF does not stimulate neurite extension. We report that EGF, in combination with KCl, stimulates neurite outgrowth in PC12 cells. This phenomenon was independent of intracellular Ca2+ increases and not due to enhancement of MAP kinase activity over that seen with EGF alone. However, EGF plus KCl increased intracellular cAMP, and other cAMP elevating agents acted synergistically with EGF to promote neurite outgrowth. Stimulation of neurite outgrowth by cAMP and EGF was blocked by inhibitors of transcription suggesting that synergistic regulation of transcription by the cAMP and MAP kinase pathways may stimulate neurite growth.
Asunto(s)
Factor de Crecimiento Epidérmico/farmacología , Neuritas/fisiología , Cloruro de Potasio/farmacología , Transducción de Señal , Animales , Calcio/metabolismo , Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , Diferenciación Celular , AMP Cíclico/metabolismo , Sinergismo Farmacológico , Quinasas de Proteína Quinasa Activadas por Mitógenos , Factores de Crecimiento Nervioso/farmacología , Células PC12/efectos de los fármacos , Biosíntesis de Proteínas , Proteínas Quinasas/genética , Proteínas Quinasas/metabolismo , Ratas , Transcripción GenéticaAsunto(s)
Aneurisma/fisiopatología , Nervio Femoral/fisiopatología , Arteria Ilíaca , Enfermedades del Sistema Nervioso Periférico/fisiopatología , Aneurisma/complicaciones , Aneurisma/diagnóstico , Femenino , Humanos , Persona de Mediana Edad , Músculos/fisiopatología , Enfermedades del Sistema Nervioso Periférico/etiología , RoturaRESUMEN
Involvement of both jejunum and ileum is uncommon in Crohn's disease of the small bowel. We report five patients with multiple strictures of the small bowel associated with one or more intervening segments of dilated bowel. A diagnosis of Crohn's disease was delayed because none of the patients experienced diarrhea. Despite the early radiologic appearance of extensive small bowel disease, only three patients have required surgery, a limited surgical resection of 65-75 cm was possible, and long-term prognosis has been favorable.