RESUMEN
ATP synthase (F1Fo) is a rotary molecular engine that harnesses energy from electrochemical-gradients across the inner mitochondrial membrane for ATP synthesis. Despite the accepted tenet that F1Fo transports exclusively H+, our laboratory has demonstrated that, in addition to H+, F1Fo ATP synthase transports a significant fraction of ΔΨm-driven charge as K+ to synthesize ATP. Herein, we utilize a computational modeling approach as a proof of principle of the feasibility of the core mechanism underlying the enhanced ATP synthesis, and to explore its bioenergetic consequences. A minimal model comprising the 'core' mechanism constituted by ATP synthase, driven by both proton (PMF) and potassium motive force (KMF), respiratory chain, adenine nucleotide translocator, Pi carrier, and K+/H+ exchanger (KHEmito) was able to simulate enhanced ATP synthesis and respiratory fluxes determined experimentally with isolated heart mitochondria. This capacity of F1Fo ATP synthase confers mitochondria with a significant energetic advantage compared to K+ transport through a channel not linked to oxidative phosphorylation (OxPhos). The K+-cycling mechanism requires a KHEmito that exchanges matrix K+ for intermembrane space H+, leaving PMF as the overall driving energy of OxPhos, in full agreement with the standard chemiosmotic mechanism. Experimental data of state 4â3 energetic transitions, mimicking low to high energy demand, could be reproduced by an integrated computational model of mitochondrial function that incorporates the 'core' mechanism. Model simulations display similar behavior compared to the experimentally observed changes in ΔΨm, mitochondrial K+ uptake, matrix volume, respiration, and ATP synthesis during the energetic transitions at physiological pH and K+ concentration. The model also explores the role played by KHEmito in modulating the energetic performance of mitochondria. The results obtained support the available experimental evidence on ATP synthesis driven by K+ and H+ transport through the F1Fo ATP synthase.
Asunto(s)
Membranas Mitocondriales , Potasio/metabolismo , Protones , Adenosina Trifosfato , Simulación por Computador , Mitocondrias Cardíacas/metabolismo , Membranas Mitocondriales/metabolismo , ATPasas de Translocación de Protón Mitocondriales/metabolismoRESUMEN
Constitutive Ca(2+)/calmodulin (CaM)-activation of adenylyl cyclases (ACs) types 1 and 8 in sinoatrial nodal cells (SANC) generates cAMP within lipid-raft-rich microdomains to initiate cAMP-protein kinase A (PKA) signaling, that regulates basal state rhythmic action potential firing of these cells. Mounting evidence in other cell types points to a balance between Ca(2+)-activated counteracting enzymes, ACs and phosphodiesterases (PDEs) within these cells. We hypothesized that the expression and activity of Ca(2+)/CaM-activated PDE Type 1A is higher in SANC than in other cardiac cell types. We found that PDE1A protein expression was 5-fold higher in sinoatrial nodal tissue than in left ventricle, and its mRNA expression was 12-fold greater in the corresponding isolated cells. PDE1 activity (nimodipine-sensitive) accounted for 39% of the total PDE activity in SANC lysates, compared to only 4% in left ventricular cardiomyocytes (LVC). Additionally, total PDE activity in SANC lysates was lowest (10%) in lipid-raft-rich and highest (76%) in lipid-raft-poor fractions (equilibrium sedimentation on a sucrose density gradient). In intact cells PDE1A immunolabeling was not localized to the cell surface membrane (structured illumination microscopy imaging), but located approximately within about 150nm inside of immunolabeling of hyperpolarization-activated cyclic nucleotide-gated potassium channels (HCN4), which reside within lipid-raft-rich microenvironments. In permeabilized SANC, in which surface membrane ion channels are not functional, nimodipine increased spontaneous SR Ca(2+) cycling. PDE1A mRNA silencing in HL-1 cells increased the spontaneous beating rate, reduced the cAMP, and increased cGMP levels in response to IBMX, a broad spectrum PDE inhibitor (detected via fluorescence resonance energy transfer microscopy). We conclude that signaling via cAMP generated by Ca(2+)/CaM-activated AC in SANC lipid raft domains is limited by cAMP degradation by Ca(2+)/CaM-activated PDE1A in non-lipid raft domains. This suggests that local gradients of [Ca(2+)]-CaM or different AC and PDE1A affinity regulate both cAMP production and its degradation, and this balance determines the intensity of Ca(2+)-AC-cAMP-PKA signaling that drives SANC pacemaker function.
Asunto(s)
Fosfodiesterasas de Nucleótidos Cíclicos Tipo 1/genética , Expresión Génica , Sistema de Conducción Cardíaco , Nodo Sinoatrial/citología , Nodo Sinoatrial/metabolismo , Animales , Calcio/metabolismo , Calmodulina/metabolismo , Línea Celular , AMP Cíclico/metabolismo , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 1/metabolismo , Activación Enzimática , Activación del Canal Iónico , Mitocondrias , Modelos Biológicos , Miocitos Cardíacos/metabolismo , Especificidad de Órganos/genética , Conejos , Canal Liberador de Calcio Receptor de Rianodina/metabolismo , Retículo Sarcoplasmático/metabolismo , Transducción de SeñalRESUMEN
Voltage-gated Ca(v)1.2 calcium channels couple membrane depolarization to cAMP response-element-binding protein (CREB)-dependent transcriptional activation. To investigate the spatial and temporal organization of CREB-dependent transcriptional nuclear microdomains, we combined perforated patch-clamp technique and FRET microscopy for monitoring CREB and CREB-binding protein interaction in the nuclei of live cells. The experimental approach to the quantitative assessment of CREB-dependent transcriptional signaling evoked by cAMP- and Ca(v)1.2-dependent mechanisms was devised in COS1 cells expressing recombinant Ca(v)1.2 calcium channels. Using continuous 2-dimensional wavelet transform and time series analyses, we found that nuclear CREB-dependent transcriptional signaling is organized differentially in spatially and temporally separated microdomains of 4 distinct types. In rat neonatal cardiomyocytes, CREB-dependent transcription is mediated by the cAMP-initiated CaMKII-sensitive and Ca(v)1.2-initiated CaMKII-insensitive mechanisms. The latter microdomains show a tendency to exhibit periodic behavior correlated with spontaneous contraction of myocytes suggestive of frequency-dependent CREB-dependent transcriptional regulation in the heart.
Asunto(s)
Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Animales , Animales Recién Nacidos , Células COS , Células Cultivadas , Chlorocebus aethiops , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/genética , Electrofisiología , Modelos Teóricos , Miocitos Cardíacos/metabolismo , Ratas , Ratas Endogámicas F344 , Transducción de Señal/genética , Transducción de Señal/fisiología , Transcripción Genética/genéticaRESUMEN
ATP synthase (F1Fo) synthesizes daily our body's weight in ATP, whose production-rate can be transiently increased several-fold to meet changes in energy utilization. Using purified mammalian F1Fo-reconstituted proteoliposomes and isolated mitochondria, we show F1Fo can utilize both ΔΨm-driven H+- and K+-transport to synthesize ATP under physiological pH = 7.2 and K+ = 140 mEq/L conditions. Purely K+-driven ATP synthesis from single F1Fo molecules measured by bioluminescence photon detection could be directly demonstrated along with simultaneous measurements of unitary K+ currents by voltage clamp, both blocked by specific Fo inhibitors. In the presence of K+, compared to osmotically-matched conditions in which this cation is absent, isolated mitochondria display 3.5-fold higher rates of ATP synthesis, at the expense of 2.6-fold higher rates of oxygen consumption, these fluxes being driven by a 2.7:1 K+: H+ stoichiometry. The excellent agreement between the functional data obtained from purified F1Fo single molecule experiments and ATP synthase studied in the intact mitochondrion under unaltered OxPhos coupling by K+ presence, is entirely consistent with K+ transport through the ATP synthase driving the observed increase in ATP synthesis. Thus, both K+ (harnessing ΔΨm) and H+ (harnessing its chemical potential energy, ΔµH) drive ATP generation during normal physiology.
Asunto(s)
Adenosina Trifosfato , ATPasas de Translocación de Protón Mitocondriales , Animales , ATPasas de Translocación de Protón Mitocondriales/química , Adenosina Trifosfato/metabolismo , Mitocondrias/metabolismo , Consumo de Oxígeno , Mamíferos/metabolismoRESUMEN
We demonstrated that ATP synthase serves the functions of a primary mitochondrial K+ "uniporter," i.e., the primary way for K+ to enter mitochondria. This K+ entry is proportional to ATP synthesis, regulating matrix volume and energy supply-vs-demand matching. We show that ATP synthase can be upregulated by endogenous survival-related proteins via IF1. We identified a conserved BH3-like domain of IF1 which overlaps its "minimal inhibitory domain" that binds to the ß-subunit of F1. Bcl-xL and Mcl-1 possess a BH3-binding-groove that can engage IF1 and exert effects, requiring this interaction, comparable to diazoxide to augment ATP synthase's H+ and K+ flux and ATP synthesis. Bcl-xL and Mcl-1, but not Bcl-2, serve as endogenous regulatory ligands of ATP synthase via interaction with IF1 at this BH3-like domain, to increase its chemo-mechanical efficiency, enabling its function as the recruitable mitochondrial KATP-channel that can limit ischemia-reperfusion injury. Using Bayesian phylogenetic analysis to examine potential bacterial IF1-progenitors, we found that IF1 is likely an ancient (â¼2 Gya) Bcl-family member that evolved from primordial bacteria resident in eukaryotes, corresponding to their putative emergence as symbiotic mitochondria, and functioning to prevent their parasitic ATP consumption inside the host cell.
Asunto(s)
Mitocondrias , ATPasas de Translocación de Protón Mitocondriales , Teorema de Bayes , Proteína 1 de la Secuencia de Leucemia de Células Mieloides/metabolismo , Filogenia , ATPasas de Translocación de Protón Mitocondriales/genética , Mitocondrias/metabolismo , Adenosina Trifosfato/metabolismoRESUMEN
Voltage-gated calcium channels conduct Ca(2+) ions in response to membrane depolarization. The resulting transient increase in cytoplasmic free calcium concentration is a critical trigger for the initiation of such vital responses as muscle contraction and transcription. L-type Ca(v)1.2 calcium channels are complexes of the pore-forming α(1C) subunit associated with cytosolic Ca(v)ß subunits. All major Ca(v)ßs share a highly homologous membrane associated guanylate kinase-like (MAGUK) domain that binds to α(1C) at the α-interaction domain (AID), a short motif in the linker between transmembrane repeats I and II. In this study we show that Ca(v)ß subunits form multimolecular homo- and heterooligomeric complexes in human vascular smooth muscle cells expressing native calcium channels and in Cos7 cells expressing recombinant Ca(v)1.2 channel subunits. Ca(v)ßs oligomerize at the α(1C) subunits residing in the plasma membrane and bind to the AID. However, Ca(v)ß oligomerization occurs independently on the association with α(1C). Molecular structures responsible for Ca(v)ß oligomerization reside in 3 regions of the guanylate kinase subdomain of MAGUK. An augmentation of Ca(v)ß homooligomerization significantly increases the calcium current density, while heterooligomerization may also change the voltage-dependence and inactivation kinetics of the channel. Thus, oligomerization of Ca(v)ß subunits represents a novel and essential aspect of calcium channel regulation.
Asunto(s)
Canales de Calcio Tipo L/metabolismo , Calcio/metabolismo , Subunidades de Proteína/metabolismo , Western Blotting , Canales de Calcio Tipo L/genética , Células Cultivadas , Electrofisiología , Transferencia Resonante de Energía de Fluorescencia , Humanos , Inmunoprecipitación , Microscopía Confocal , Multimerización de Proteína/genética , Multimerización de Proteína/fisiología , Subunidades de Proteína/genéticaRESUMEN
It is generally accepted that to generate calcium currents in response to depolarization, Ca(v)1.2 calcium channels require association of the pore-forming alpha(1C) subunit with accessory Ca(v)beta and alpha(2)delta subunits. A single calmodulin (CaM) molecule is tethered to the C-terminal alpha(1C)-LA/IQ region and mediates Ca2+-dependent inactivation of the channel. Ca(v)beta subunits are stably associated with the alpha(1C)-interaction domain site of the cytoplasmic linker between internal repeats I and II and also interact dynamically, in a Ca2+-dependent manner, with the alpha(1C)-IQ region. Here, we describe a surprising discovery that coexpression of exogenous CaM (CaM(ex)) with alpha(1C)/alpha(2)delta in COS1 cells in the absence of Ca(v)beta subunits stimulates the plasma membrane targeting of alpha(1C), facilitates calcium channel gating, and supports Ca2+-dependent inactivation. Neither real-time PCR with primers complementary to monkey Ca(v)beta subunits nor coimmunoprecipitation analysis with exogenous alpha(1C) revealed an induction of endogenous Ca(v)beta subunits that could be linked to the effect of CaM(ex). Coexpression of a calcium-insensitive CaM mutant CaM(1234) also facilitated gating of Ca(v)beta-free Ca(v)1.2 channels but did not support Ca2+-dependent inactivation. Our results show there is a functional matchup between CaM(ex) and Ca(v)beta subunits that, in the absence of Ca(v)beta, renders Ca2+ channel gating facilitated by CaM molecules other than the one tethered to LA/IQ to support Ca2+-dependent inactivation. Thus, coexpression of CaM(ex) creates conditions when the channel gating, voltage- and Ca2+-dependent inactivation, and plasma-membrane targeting occur in the absence of Ca(v)beta. We suggest that CaM(ex) affects specific Ca(v)beta-free conformations of the channel that are not available to endogenous CaM.
Asunto(s)
Canales de Calcio Tipo L/metabolismo , Calmodulina/metabolismo , Activación del Canal Iónico , Subunidades de Proteína/metabolismo , Animales , Células COS , Calcio/farmacología , Calmodulina/farmacología , Chlorocebus aethiops , Electrofisiología , Humanos , Activación del Canal Iónico/efectos de los fármacos , Proteínas Mutantes/metabolismo , Unión Proteica/efectos de los fármacos , Proteínas Recombinantes/metabolismoRESUMEN
Spontaneous firing of sinoatrial (SA) node cells (SANCs) is regulated by cyclic adenosine monophosphate (cAMP)-mediated, protein kinase A (PKA)-dependent (cAMP/PKA) local subsarcolemmal Ca2+ releases (LCRs) from ryanodine receptors (RyR). The LCRs occur during diastolic depolarization (DD) and activate an inward Na+/Ca2+ exchange current that accelerates the DD rate prompting the next action potential (AP). Basal phosphodiesterases (PDEs) activation degrades cAMP, reduces basal cAMP/PKA-dependent phosphorylation, and suppresses normal spontaneous firing of SANCs. The cAMP-degrading PDE1, PDE3, and PDE4 represent major PDE activities in rabbit SANC, and PDE inhibition by 3-isobutyl-1-methylxanthine (IBMX) increases spontaneous firing of SANC by â¼50%. Though inhibition of single PDE1-PDE4 only moderately increases spontaneous SANC firing, dual PDE3 + PDE4 inhibition produces a synergistic effect hastening the spontaneous SANC beating rate by â¼50%. Here, we describe the expression and distribution of different PDE subtypes within rabbit SANCs, several specific targets (L-type Ca2+ channels and phospholamban) regulated by basal concurrent PDE3 + PDE4 activation, and critical importance of RyR Ca2+ releases for PDE-dependent regulation of spontaneous SANC firing. Colocalization of PDE3 and PDE4 beneath sarcolemma or in striated patterns inside SANCs strongly suggests that PDE-dependent regulation of cAMP/PKA signaling might be executed at the local level; this idea, however, requires further verification.
RESUMEN
Fluorescence microscopy imaging has become one of the most useful techniques to assess the activity of individual cells, subcellular trafficking of signals to and between organelles, and to appreciate how organelle function is regulated. The past 2 decades have seen a tremendous advance in the rational design and development in the nature and selectivity of probes to serve as reporters of the intracellular environment in live cells. These probes range from small organic fluorescent molecules to fluorescent biomolecules and photoproteins ingeniously engineered to follow signaling traffic, sense ionic and nonionic second messengers, and report various kinase activities. These probes, together with recent advances in imaging technology, have enabled significantly enhanced spatial and temporal resolution. This review summarizes some of these developments and their applications to assess intracellular organelle function.
Asunto(s)
Colorantes Fluorescentes/análisis , Microscopía Fluorescente/métodos , Orgánulos/fisiología , Animales , Núcleo Celular/metabolismo , Núcleo Celular/ultraestructura , Retículo Endoplásmico/metabolismo , Retículo Endoplásmico/ultraestructura , Transferencia Resonante de Energía de Fluorescencia , Colorantes Fluorescentes/química , Colorantes Fluorescentes/farmacocinética , Genes Reporteros , Humanos , Mediciones Luminiscentes , Proteínas Luminiscentes/análisis , Proteínas Luminiscentes/química , Proteínas Luminiscentes/genética , Mitocondrias/química , Mitocondrias/fisiología , Mitocondrias/ultraestructura , Nanotecnología , Orgánulos/química , Orgánulos/ultraestructura , Oxidación-Reducción , Fotoblanqueo , Puntos Cuánticos , Retículo Sarcoplasmático/metabolismo , Retículo Sarcoplasmático/ultraestructura , Sistemas de Mensajero SecundarioRESUMEN
The major function of the voltage-gated calcium channels is to provide the Ca(2+) flux into the cell. L-type voltage-gated calcium channels (Cav1) serve as voltage sensors that couple membrane depolarization to many intracellular processes. Electrical activity in excitable cells affects gene expression through signaling pathways involved in the excitation-transcription (E-T) coupling. E-T coupling starts with activation of the Cav1 channel and results in initiation of the cAMP-response element binding protein (CREB)-dependent transcription. In this review we discuss the new quantitative approaches to measuring E-T signaling events. We describe the use of wavelet transform to detect heterogeneity of transcriptional activation in nuclei. Furthermore, we discuss the properties of discovered microdomains of nuclear signaling associated with the E-T coupling and the basis of the frequency-dependent transcriptional regulation.
Asunto(s)
Canales de Calcio/metabolismo , AMP Cíclico/metabolismo , Activación Transcripcional , Animales , HumanosRESUMEN
Fluorophore-assisted light inactivation (FALI) is an investigative tool to inactivate fluorescently labeled proteins by a mechanism of in situ photodestruction. We found that Ca(v)1.2 (L-type) and Ca(v)3.1 (T-type) calcium channels, labeled by genetic fusion with GFP derivatives, show differential sensitivity to FALI. Specifically, FALI silences Ca(v)1.2 calcium channels containing EYFP-labeled α(1C)subunits but does not affect the EYFP-α(1G) Ca(v)3.1 calcium channels or Ca(v)1.2 channels containing EYFP-labeled ß subunits. Our findings limit the applicability of acceptor photobleaching for the measurements of FRET but open an opportunity to combine the fluorescent imaging of the live cell expressing labeled calcium channels with selective functional inactivation of their specific subsets.
Asunto(s)
Canales de Calcio Tipo L/metabolismo , Canales de Calcio Tipo T/metabolismo , Inactivación por Luz Asistida por Cromóforo , Animales , Células COS , Canales de Calcio Tipo L/genética , Canales de Calcio Tipo T/genética , Chlorocebus aethiops , Transferencia Resonante de Energía de Fluorescencia/métodos , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Activación del Canal Iónico , Microscopía Fluorescente/métodos , Fotoblanqueo , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Rayos UltravioletaAsunto(s)
Carbunco/metabolismo , Antígenos Bacterianos/metabolismo , Toxinas Bacterianas/metabolismo , Canales de Calcio/metabolismo , Sitios de Unión , Calcio/metabolismo , Línea Celular , Fenómenos Electrofisiológicos , Potenciales de la Membrana , Unión Proteica , Conformación Proteica , Transducción de SeñalRESUMEN
Voltage-activated CaV1.2 calcium channels require association of the pore-forming alpha1C subunit with accessory CaVbeta and alpha2delta subunits. Binding of a single calmodulin (CaM) to alpha1C supports Ca2+-dependent inactivation (CDI). The human CaV1.2 channel is silent in the absence of CaVbeta and/or alpha2delta. Recently, we found that coexpression of exogenous CaM (CaMex) supports plasma membrane targeting, gating facilitation and CDI of the channel in the absence of CaVbeta. Here we discovered that CaMex and its Ca2+-insensitive mutant (CaM1234) rendered active alpha1C/CaVbeta channel in the absence of alpha2delta. Coexpression of CaMex with alpha1C and beta2d in calcium-channel-free COS-1 cells recovered gating of the channel and supported CDI. Voltage-dependence of activation was shifted by approximately +40 mV to depolarization potentials. The calcium current reached maximum at +40 mV (20 mM Ca2+) and exhibited approximately 3 times slower activation and 5 times slower inactivation kinetics compared to the wild-type channel. Furthermore, both CaMex and CaM1234 accelerated recovery from inactivation and induced facilitation of the calcium current by strong depolarization prepulse, the properties absent from the human vascular/neuronal CaV1.2 channel. The data suggest a previously unknown action of CaM that in the presence of CaVbeta; translates into activation of the alpha2delta-deficient calcium channel and alteration of its properties.
Asunto(s)
Canales de Calcio Tipo L/metabolismo , Canales de Calcio/metabolismo , Calcio/metabolismo , Calmodulina/metabolismo , Activación del Canal Iónico , Animales , Células COS , Canales de Calcio/genética , Canales de Calcio Tipo L/química , Canales de Calcio Tipo L/genética , Calmodulina/genética , Chlorocebus aethiops , Humanos , Potenciales de la Membrana , Modelos Biológicos , Mutación , Factores de Tiempo , TransfecciónRESUMEN
BACKGROUND: Voltage-gated Ca(v)1.2 calcium channels play a crucial role in Ca(2+) signaling. The pore-forming alpha(1C) subunit is regulated by accessory Ca(v)beta subunits, cytoplasmic proteins of various size encoded by four different genes (Ca(v)beta(1)-beta(4)) and expressed in a tissue-specific manner. METHODS AND RESULTS: Here we investigated the effect of three major Ca(v)beta types, beta(1b), beta(2d) and beta(3), on the structure of Ca(v)1.2 in the plasma membrane of live cells. Total internal reflection fluorescence microscopy showed that the tendency of Ca(v)1.2 to form clusters depends on the type of the Ca(v)beta subunit present. The highest density of Ca(v)1.2 clusters in the plasma membrane and the smallest cluster size were observed with neuronal/cardiac beta(1b) present. Ca(v)1.2 channels containing beta(3), the predominant Ca(v)beta subunit of vascular smooth muscle cells, were organized in a significantly smaller number of larger clusters. The inter- and intramolecular distances between alpha(1C) and Ca(v)beta in the plasma membrane of live cells were measured by three-color FRET microscopy. The results confirm that the proximity of Ca(v)1.2 channels in the plasma membrane depends on the Ca(v)beta type. The presence of different Ca(v)beta subunits does not result in significant differences in the intramolecular distance between the termini of alpha(1C), but significantly affects the distance between the termini of neighbor alpha(1C) subunits, which varies from 67 A with beta(1b) to 79 A with beta(3). CONCLUSIONS: Thus, our results show that the structural organization of Ca(v)1.2 channels in the plasma membrane depends on the type of Ca(v)beta subunits present.
Asunto(s)
Canales de Calcio Tipo L/química , Canales de Calcio Tipo N/metabolismo , Subunidades de Proteína/metabolismo , Canales de Calcio Tipo L/metabolismo , Canales de Calcio Tipo N/química , Transferencia Resonante de Energía de Fluorescencia , Humanos , Proteínas Luminiscentes/metabolismo , Subunidades de Proteína/química , Proteína Fluorescente RojaRESUMEN
Ca(v)beta subunits support voltage gating of Ca(v)1.2 calcium channels and play important role in excitation-contraction coupling. The common central membrane-associated guanylate kinase (MAGUK) region of Ca(v)beta binds to the alpha-interaction domain (AID) and the IQ motif of the pore-forming alpha(1C) subunit, but these two interactions do not explain why the cardiac Ca(v)beta(2) subunit splice variants differentially modulate inactivation of Ca(2+) currents (I(Ca)). Previously we described beta(2Deltag), a functionally active splice variant of human Ca(v)beta(2) lacking MAGUK. By deletion analysis of beta(2Deltag), we have now identified a 41-amino acid C-terminal essential determinant (beta(2)CED) that stimulates I(Ca) in the absence of Ca(v)beta subunits and conveys a +20-mV shift in the peak of the I(Ca)-voltage relationship. The beta(2)CED is targeted by alpha(1C) to the plasma membrane, forms a complex with alpha(1C) but does not bind to AID. Electrophysiology and binding studies point to the calmodulin-interacting LA/IQ region in the alpha(1C) subunit C terminus as a functionally relevant beta(2)CED binding site. The beta(2)CED interacts with LA/IQ in a Ca(2+)- and calmodulin-independent manner and need LA, but not IQ, to activate the channel. Deletion/mutation analyses indicated that each of the three Ca(v)beta(2)/alpha(1C) interactions is sufficient to support I(Ca). However, beta(2)CED does not support Ca(2+)-dependent inactivation, suggesting that interactions of MAGUK with AID and IQ are crucial for Ca(2+)-induced inactivation. The beta(2)CED is conserved only in Ca(v)beta(2) subunits. Thus, beta(2)CED constitutes a previously unknown integrative part of the multifactorial mechanism of Ca(v)beta(2)-subunit differential modulation of the Ca(v)1.2 calcium channel that in beta(2Deltag) occurs without MAGUK.
Asunto(s)
Canales de Calcio Tipo L/metabolismo , Calcio/metabolismo , Membrana Celular/metabolismo , Activación del Canal Iónico/fisiología , Potenciales de la Membrana/fisiología , Subunidades de Proteína/metabolismo , Secuencias de Aminoácidos/fisiología , Secuencia de Aminoácidos/genética , Sitios de Unión/fisiología , Canales de Calcio Tipo L/genética , Calmodulina/genética , Calmodulina/metabolismo , Línea Celular , Membrana Celular/genética , Humanos , Miocardio/metabolismo , Estructura Terciaria de Proteína/fisiología , Subunidades de Proteína/genética , Eliminación de SecuenciaRESUMEN
A technique that utilizes the one-dimensional (1D) continuous wavelet transform (CWT) of linearized fluorescence resonance energy transfer (FRET) microscopic images has been extended to identify signaling macro- and microdomains in cell plasma membranes by incorporating the two-dimensional (2D) CWT of time-lapse fluorescence and/or FRET images. Signaling domains were identified from differences in wavelet coefficient matrices, and there was good agreement between the 1D and 2D methods on examining a), static fluorescent images of COS1 cells expressing calmodulin kinase II fused with enhanced yellow fluorescent protein, and b), time lapse FRET images of reporters of protein kinase C (PKC) (PKC activity reporter) and adenylyl cyclase dynamics (cAMP) activity within COS1 plasma membrane confines after stimulation by phorbol-12,13-dibutyrate or forskolin, respectively. The proposed 2D wavelet-based image analysis effectively detected phosphorylation/dephosphorylation signaling microdomains (PKC) as well as those reflective of cAMP without the limitation of requiring linearized signals imposed by the 1D approach. Illustrating successful application to the analysis of intracellular compartments, the 2D CWT was further used to identify signaling domains of cAMP response element-binding (CREB)-induced transcriptional activation in the nuclei of COS1 cells, which could not be achieved with the 1D approach. This technique may be eventually used to characterize complex cellular signaling and protein-protein interactions within localized cytoplasmic domains.
Asunto(s)
Algoritmos , Transferencia Resonante de Energía de Fluorescencia/métodos , Interpretación de Imagen Asistida por Computador/métodos , Proteínas de la Membrana/metabolismo , Microscopía Fluorescente/métodos , Transducción de Señal/fisiología , Animales , Células COS , Chlorocebus aethiops , Estructura Terciaria de ProteínaRESUMEN
The voltage-gated Kv2.1 channel is composed of four identical subunits folded around the central pore and does not inactivate appreciably during short depolarizing pulses. To study voltage-induced relative molecular rearrangements of the channel, Kv2.1 subunits were genetically fused with enhanced cyan fluorescent protein and/or enhanced yellow fluorescent protein, expressed in COS1 cells, and investigated using fluorescence resonance energy transfer (FRET) microscopy combined with patch clamp. Fusion of fluorophores to either or both termini of the Kv2.1 monomer did not significantly affect the gating properties of the channel. FRET between the N- and C-terminal tags fused to the same or different Kv2.1 monomers decreased upon activation of the channel by depolarization from -80 to +60 mV, suggesting voltage-gated relative rearrangement between the termini. Because FRET between the Kv2.1 N- or C-terminal tags and the membrane-trapped EYFP(N)-PH pleckstrin homology domains did not change on depolarization, voltage-gated relative movements between the Kv2.1 termini occurred in a plane parallel to the plasma membrane, within a distance of 1-10 nm. FRET between the N-terminal tags did not change upon depolarization, indicating that the N termini do not rearrange relative to each other, but they could either move cooperatively with the Kv2.1 tetramer or not move at all. No FRET was detected between the C-terminal tags. Assuming their randomized orientation in the symmetrically arranged Kv2.1 subunits, C termini may move outwards in order to produce relative rearrangements between N and C termini upon depolarization.
Asunto(s)
Canales de Potasio Shab/química , Canales de Potasio Shab/fisiología , Animales , Proteínas Sanguíneas/química , Células COS , Membrana Celular/metabolismo , Chlorocebus aethiops , Electrofisiología , Transferencia Resonante de Energía de Fluorescencia , Modelos Estadísticos , Técnicas de Placa-Clamp , Fosfoproteínas/química , Estructura Terciaria de Proteína , Factores de Tiempo , TransfecciónRESUMEN
In this study, we sought to characterize functional signaling domains by applying the multiresolution properties of the continuous wavelet transform to fluorescence resonance energy transfer (FRET) microscopic images of plasma membranes. A genetically encoded FRET reporter of protein kinase C (PKC)-dependent phosphorylation was expressed in COS1 cells. Differences between wavelet coefficient matrices revealed several heterogeneous domains (typically ranging from 1 to 5 microm), reflecting the dynamic balance between PKC and phosphatase activity during stimulation with phorbol-12,13-dibutyrate or acetylcholine. The balance in these domains was not necessarily reflected in the overall plasma membrane changes, and observed heterogeneity was absent when cells were exposed to a phosphatase or PKC inhibitor. Prolonged exposure to phorbol-12,13-dibutyrate and acetylcholine yielded more homogeneous FRET distribution in plasma membranes. The proposed wavelet-based image analysis provides, for the first time, a basis and a means of detecting and quantifying dynamic changes in functional signaling domains, and may find broader application in studying fine aspects of cellular signaling by various imaging reporters.
Asunto(s)
Membrana Celular/metabolismo , Transferencia Resonante de Energía de Fluorescencia/métodos , Proteínas Adaptadoras Transductoras de Señales/química , Animales , Proteínas Bacterianas/química , Células COS , Genes Reporteros , Proteínas Fluorescentes Verdes/química , Procesamiento de Imagen Asistido por Computador , Proteínas Luminiscentes/química , Modelos Estadísticos , Forbol 12,13-Dibutirato/química , Monoéster Fosfórico Hidrolasas/metabolismo , Fosforilación , Proteína Quinasa C/química , Proteína Quinasa C/genética , Estructura Terciaria de Proteína , Transducción de Señal , Factores de TiempoRESUMEN
Voltage-gated Ca(v)1.2 channels are composed of the pore-forming alpha1C and auxiliary beta and alpha2delta subunits. Voltage-dependent conformational rearrangements of the alpha1C subunit C-tail have been implicated in Ca2+ signal transduction. In contrast, the alpha1C N-tail demonstrates limited voltage-gated mobility. We have asked whether these properties are critical for the channel function. Here we report that transient anchoring of the alpha1C subunit C-tail in the plasma membrane inhibits Ca2+-dependent and slow voltage-dependent inactivation. Both alpha2delta and beta subunits remain essential for the functional channel. In contrast, if alpha1C subunits with are expressed alpha2delta but in the absence of a beta subunit, plasma membrane anchoring of the alpha1C N terminus or its deletion inhibit both voltage- and Ca2+-dependent inactivation of the current. The following findings all corroborate the importance of the alpha1C N-tail/beta interaction: (i) co-expression of beta restores inactivation properties, (ii) release of the alpha1C N terminus inhibits the beta-deficient channel, and (iii) voltage-gated mobility of the alpha1C N-tail vis a vis the plasma membrane is increased in the beta-deficient (silent) channel. Together, these data argue that both the alpha1C N- and C-tails have important but different roles in the voltage- and Ca2+-dependent inactivation, as well as beta subunit modulation of the channel. The alpha1C N-tail may have a role in the channel trafficking and is a target of the beta subunit modulation. The beta subunit facilitates voltage gating by competing with the N-tail and constraining its voltage-dependent rearrangements. Thus, cross-talk between the alpha1C C and N termini, beta subunit, and the cytoplasmic pore region confers the multifactorial regulation of Ca(v)1.2 channels.