共查询到20条相似文献,搜索用时 15 毫秒
1.
Michael L. Woodruff Ammaji Rajala Gordon L. Fain Raju V. S. Rajala 《The Journal of biological chemistry》2014,289(1):358-364
Previous experiments have indicated that growth factor receptor-bound protein 14 (Grb14) may modulate rod photoreceptor cGMP-gated channels by decreasing channel affinity for cGMP; however, the function of Grb14 in rod physiology is not known. In this study, we examined the role of Grb14 by recording electrical responses from rods in which the gene for the Grb14 protein had been deleted. Suction-electrode recordings from single mouse rods showed that responses of dark-adapted Grb14−/− mice to brief flashes decayed more rapidly than strain-controlled wild type (WT) rods, with decreased values of both integration time and the exponential time course of decay (τREC). This result is consistent with an increase in channel affinity for cGMP produced by deletion of Grb14. However, Grb14−/− mouse rods also showed little change in dark current and a large and significant decrease in the limiting time constant τD, which are not consistent with an effect on channel affinity but seem rather to indicate modulation of the rate of inactivation of cyclic nucleotide phosphodiesterase 6 (PDE6). Grb14 has been reported to translocate from the inner to the outer segment in bright light, but we saw effects on response time course even in dark-adapted rods, although the effects were somewhat greater after rods had been adapted by exposure to bleaching illumination. Our results indicate that the mechanism of Grb14 action may be more complex than previously realized. 相似文献
2.
Keisuke Sakurai Jeannie Chen Shahrokh C. Khani Vladimir J. Kefalov 《The Journal of biological chemistry》2015,290(14):9239-9250
Cone photoreceptors function under daylight conditions and are essential for color perception and vision with high temporal and spatial resolution. A remarkable feature of cones is that, unlike rods, they remain responsive in bright light. In rods, light triggers a decline in intracellular calcium, which exerts a well studied negative feedback on phototransduction that includes calcium-dependent inhibition of rhodopsin kinase (GRK1) by recoverin. Rods and cones share the same isoforms of recoverin and GRK1, and photoactivation also triggers a calcium decline in cones. However, the molecular mechanisms by which calcium exerts negative feedback on cone phototransduction through recoverin and GRK1 are not well understood. Here, we examined this question using mice expressing various levels of GRK1 or lacking recoverin. We show that although GRK1 is required for the timely inactivation of mouse cone photoresponse, gradually increasing its expression progressively delays the cone response recovery. This surprising result is in contrast with the known effect of increasing GRK1 expression in rods. Notably, the kinetics of cone responses converge and become independent of GRK1 levels for flashes activating more than ∼1% of cone pigment. Thus, mouse cone response recovery in bright light is independent of pigment phosphorylation and likely reflects the spontaneous decay of photoactivated visual pigment. We also find that recoverin potentiates the sensitivity of cones in dim light conditions but does not contribute to their capacity to function in bright light. 相似文献
3.
Background
The rate-limiting step that determines the dominant time constant (τD) of mammalian rod photoresponse recovery is the deactivation of the active phosphodiesterase (PDE6). Physiologically relevant Ca2+-dependent mechanisms that would affect the PDE inactivation have not been identified. However, recently it has been shown that τD is modulated by background light in mouse rods.Methodology/Principal Findings
We used ex vivo ERG technique to record pharmacologically isolated photoreceptor responses (fast PIII component). We show a novel static effect of calcium on mouse rod phototransduction: Ca2+ shortens the dominant time constant (τD) of saturated photoresponse recovery, i.e., when extracellular free Ca2+ is decreased from 1 mM to ∼25 nM, the τD is reversibly increased ∼1.5–2-fold.Conclusions
We conclude that the increase in τD during low Ca2+ treatment is not due to increased [cGMP], increased [Na+] or decreased [ATP] in rod outer segment (ROS). Also it cannot be due to protein translocation mechanisms. We suggest that a Ca2+-dependent mechanism controls the life time of active PDE. 相似文献4.
Yuki Koshitani Shuji Tachibanaki Satoru Kawamura 《The Journal of biological chemistry》2014,289(5):2651-2657
Cones are less light-sensitive than rods. We showed previously in carp that more light (>100-fold) is required in cones than in rods to activate 50% of cGMP phosphodiesterase (PDE). The lower effectiveness of PDE activation in carp cones is due partly to the fact that the activation rate of transducin (Tr) by light-activated visual pigment (R*) is 5-fold lower in carp cones than in rods. In this study, we tried to explain the remaining difference. First, we examined the efficiency of activation of PDE by activated Tr (Tr*). By activating PDE with known concentrations of the active (guanosine 5′-Ο-(γ-thio)triphosphate (GTPγS)-bound) form of Tr*, we found that Tr* activated PDE at a similar efficiency in rods and cones. Next, we examined the contribution of R* and Tr* lifetimes. In a comparison of PDE activation in the presence (with GTP) and absence (with GTPγS) of Tr* inactivation, PDE activation required more light (and was therefore less effective) when Tr* was inactivated in both rod and cone membranes. This is probably because inactivation of Tr* shortened its lifetime, thereby reducing the number of activated PDE molecules. The effect of Tr* inactivation was larger in cones, probably because the lifetime of Tr* is shorter in cones than in rods. The shorter lifetimes of Tr* and R* in cones seem to explain the remaining difference in the effectiveness of PDE activation between rods and cones. 相似文献
5.
Samantha J. Allen Gopal Parthasarathy Paul L. Darke Ronald E. Diehl Rachael E. Ford Dawn L. Hall Scott A. Johnson John C. Reid Keith W. Rickert Jennifer M. Shipman Stephen M. Soisson Paul Zuck Sanjeev K. Munshi Kevin J. Lumb 《The Journal of biological chemistry》2015,290(33):20360-20373
G-protein-coupled receptor (GPCR) kinases (GRKs) bind to and phosphorylate GPCRs, initiating the process of GPCR desensitization and internalization. GRK4 is implicated in the regulation of blood pressure, and three GRK4 polymorphisms (R65L, A142V, and A486V) are associated with hypertension. Here, we describe the 2.6 Å structure of human GRK4α A486V crystallized in the presence of 5′-adenylyl β,γ-imidodiphosphate. The structure of GRK4α is similar to other GRKs, although slight differences exist within the RGS homology (RH) bundle subdomain, substrate-binding site, and kinase C-tail. The RH bundle subdomain and kinase C-terminal lobe form a strikingly acidic surface, whereas the kinase N-terminal lobe and RH terminal subdomain surfaces are much more basic. In this respect, GRK4α is more similar to GRK2 than GRK6. A fully ordered kinase C-tail reveals interactions linking the C-tail with important determinants of kinase activity, including the αB helix, αD helix, and the P-loop. Autophosphorylation of wild-type GRK4α is required for full kinase activity, as indicated by a lag in phosphorylation of a peptide from the dopamine D1 receptor without ATP preincubation. In contrast, this lag is not observed in GRK4α A486V. Phosphopeptide mapping by mass spectrometry indicates an increased rate of autophosphorylation of a number of residues in GRK4α A486V relative to wild-type GRK4α, including Ser-485 in the kinase C-tail. 相似文献
6.
Wen Mao K. J. Miyagishima Yun Yao Brian Soreghan Alapakkam P. Sampath Jeannie Chen 《The Journal of biological chemistry》2013,288(8):5257-5267
The signaling cascades mediated by G protein-coupled receptors (GPCRs) exhibit a wide spectrum of spatial and temporal response properties to fulfill diverse physiological demands. However, the mechanisms that shape the signaling response of the GPCR are not well understood. In this study, we replaced cone transducin α (cTα) for rod transducin α (rTα) in rod photoreceptors of transgenic mice, which also express S opsin, to evaluate the role of Gα subtype on signal amplification from different GPCRs in the same cell; such analysis may explain functional differences between retinal rod and cone photoreceptors. We showed that ectopically expressed cTα 1) forms a heterotrimeric complex with rod Gβ1γ1, 2) substitutes equally for rTα in generating photoresponses initiated by either rhodopsin or S-cone opsin, and 3) exhibited similar light-activated translocation as endogenous rTα in rods and endogenous cTα in cones. Thus, rTα and cTα appear functionally interchangeable. Interestingly, light sensitivity appeared to correlate with the concentration of cTα when expression is reduced below 35% of normal. However, quantification of endogenous cTα concentration in cones showed a higher level to rTα in rods. Thus, reduced sensitivity in cones cannot be explained by reduced coupling efficiency between the GPCR and G protein or a lower concentration of G protein in cones versus rods. 相似文献
7.
Qin Fu Sungjin Kim Dagoberto Soto Vania De Arcangelis Lisa DiPilato Shubai Liu Bing Xu Qian Shi Jin Zhang Yang K. Xiang 《The Journal of biological chemistry》2014,289(21):14771-14781
Small-molecule, ligand-activated G protein-coupled receptors are generally thought to be rapidly desensitized within a period of minutes through receptor phosphorylation and internalization after repeated or prolonged stimulation. This transient G protein-coupled receptor activation remains at odds with many observed long-lasting cellular and physiological responses. Here, using live cell imaging of cAMP with a FRET-based biosensor and myocyte contraction assay, we show that the catecholamine-activated β1 adrenergic receptor (β1AR) continuously stimulates second messenger cAMP synthesis in primary cardiac myocytes and neurons, which lasts for more than 8 h (a decay t½ of 3.9 h) in cardiac myocytes. However, the β1AR-induced cAMP signal is counterbalanced and masked by the receptor-bound phosphodiesterase (PDE) 4D8-dependent cAMP hydrolysis. Inhibition of PDE4 activity recovers the receptor-induced cAMP signal and promotes contractile response in mouse hearts during extended periods of agonist stimulation. β1AR associates with PDE4D8 through the receptor C-terminal PDZ motif-dependent binding to synaptic-associated protein 97 (SAP97). Knockdown of SAP97 or mutation of the β1AR PDZ motif disrupts the complex and promotes sustained agonist-induced cAMP activity, PKA phosphorylation, and cardiac myocyte contraction response. Together, these findings unveil a long lasting adrenergic signal in neurons and myocytes under prolonged stimulation and an underappreciated role of PDE that is essential in classic receptor signaling desensitization and in maintaining a long lasting cAMP equilibrium for ligand-induced physiological response. 相似文献
8.
Van Anthony M. Villar John E. Jones Ines Armando Cynthia Palmes-Saloma Peiying Yu Annabelle M. Pascua Lindsay Keever Francis B. Arnaldo Zheng Wang Yingjin Luo Robin A. Felder Pedro A. Jose 《The Journal of biological chemistry》2009,284(32):21425-21434
During conditions of moderate sodium excess, the dopaminergic system regulates blood pressure and water and electrolyte balance by engendering natriuresis. Dopamine exerts its effects on dopamine receptors, including the dopamine D3 receptor. G protein-coupled receptor kinase 4 (GRK4), whose gene locus (4p16.3) is linked to essential hypertension, desensitizes the D1 receptor, another dopamine receptor. This study evaluated the role of GRK4 on D3 receptor function in human proximal tubule cells. D3 receptor co-segregated in lipid rafts and co-immunoprecipitated and co-localized in human proximal tubule cells and in proximal and distal tubules and glomeruli of kidneys of Wistar Kyoto rats. Bimolecular fluorescence complementation and confocal microscopy revealed that agonist activation of the receptor initiated the interaction between D3 receptor and GRK4 at the cell membrane and promoted it intracellularly, presumably en route to endosomal trafficking. Of the four GRK4 splice variants, GRK4-γ and GRK4-α mediated a 3- and 2-fold increase in the phosphorylation of agonist-activated D3 receptor, respectively. Inhibition of GRK activity with heparin or knockdown of GRK4 expression via RNA interference completely abolished p44/42 phosphorylation and mitogenesis induced by D3 receptor stimulation. These data demonstrate that GRK4, specifically the GRK4-γ and GRK4-α isoforms, phosphorylates the D3 receptor and is crucial for its signaling in human proximal tubule cells.During conditions of moderate sodium excess, the dopaminergic system sits at the fulcrum of homeostatic control of water and electrolyte balance and blood pressure (1, 2). Dopamine promotes natriuresis by inhibiting sodium chloride reabsorption in specific segments of the nephron. Dopamine exerts its action on dopamine receptors, which belong to the family of G protein-coupled receptors (GPCRs).2 The dopamine receptors are classified into two subtypes based on their ability to increase cAMP levels, sequence similarity, G protein coupling, and pharmacological profiles (3, 4). The D1-like dopamine receptors activate adenylyl cyclase by coupling to stimulatory Gαs/Gαolf and include the D1 (D1R) and D5 receptors (D5R). The D2-like dopamine receptors inhibit adenylyl cyclase by coupling to Gαi/Gαo and consist of the D2 (D2R), D3 (D3R), and D4 (D4R) receptors. The D3R has also been shown to couple to Gαo, Gβγ, and to the stimulatory Gαs (5, 6).The signal transduction that follows ligand occupation of a GPCR is tightly regulated to limit the specificity and extent of cellular response. GPCR-mediated signal transduction is rapidly dampened via receptor desensitization or the waning of the responsiveness of the receptor to agonist with time. Desensitization involves receptor phosphorylation and is carried out by either GPCR kinases (GRKs) or second messenger-activated kinases such as protein kinase A and protein kinase C. Homologous desensitization involves GRKs that selectively phosphorylate only agonist-activated receptors, whereas heterologous desensitization is carried out by second messenger-dependent kinases that indiscriminately phosphorylate agonist-activated receptors and those that have not been exposed to the agonist (7).The GRKs are serine/threonine protein kinases comprising seven isoforms that are grouped into three subfamilies. GRK1 and GRK7 belong to the rhodopsin kinase subfamily and are expressed exclusively in the retina (8–10). GRK2 and GRK3 phosphorylate the β-adrenergic receptor and belong to the β-adrenergic receptor kinase subfamily (11), and GRK4, GRK5, and GRK6 belong to the GRK4 subfamily. GRK4 is highly enriched in the testis and, to a lesser degree, in the kidneys (12, 13). Four splice variants of human GRK4 result from the alternative splicing of exons 2 and 15 (11). GRK4-α is considered the full-length version, whereas GRK4-β, -γ, and -δ are shortened versions of GRK4-α (14). The coding region of the GRK4 gene, whose 4p16.3 locus has been linked to essential hypertension (15, 16), contains several single nucleotide polymorphisms, including R65L, A142V, and A486V, which have been linked to essential hypertension and/or salt sensitivity in various ethnic groups (17).The D3R gene is found at 3q13.3 (18), a locus that is also linked to essential hypertension (19, 20). Sequence analysis of the D3R gene shows the presence of several single nucleotide polymorphisms, which do not correlate with either essential hypertension among Japanese (21) or with blood pressure levels and diabetic nephropathy among Finns (22). However, D3R knock-out mice develop a renin-dependent form of hypertension and fail to excrete a sodium load (23).The D3R has a long third intracellular loop that contains several putative GRK phosphorylation sites (24). A previous study evaluated the ability of GRK2 and GRK3 to phosphorylate D3R and showed that co-transfection of GRK3, but not GRK2, resulted in a weak phosphorylation of the heterologously expressed, dopamine-stimulated D3R in HEK-293 (25), a human embryonic kidney cell line. We tested the hypothesis that GRK4 is required in D3R signaling in terminally differentiated human renal proximal tubule cells (hPTCs) by determining the spatiotemporal dynamics of the interaction of D3R and GRK4 through their subfractionation in membrane microdomains and subcellular co-localization via confocal microscopy and bimolecular fluorescence complementation assay (BiFC). We also identified which of the GRK4 splice variants are involved in D3R phosphorylation and evaluated the physiological roles of GRK4 in D3R signaling in the hPTCs. We now report that D3R and GRK4 co-fractionate in lipid rafts and co-localize in both hPTCs and WYK kidneys. Moreover, D3R is phosphorylated by GRK4-γ and GRK4-α isoforms, and the absence of GRK4 impairs D3R-mediated mitogenesis and activation of p44/42 in hPTCs. 相似文献
9.
Kui Zhai Fabien Hubert Valérie Nicolas Guangju Ji Rodolphe Fischmeister Véronique Leblais 《PloS one》2012,7(10)
Background
We investigated the role of cyclic nucleotide phosphodiesterases (PDEs) in the spatiotemporal control of intracellular cAMP concentrations in rat aortic smooth muscle cells (RASMCs).Methodology/Principal Findings
The rank order of PDE families contributing to global cAMP-PDE activity was PDE4> PDE3 = PDE1. PDE7 mRNA expression but not activity was confirmed. The Fluorescence Resonance Energy Transfer (FRET)-based cAMP sensor, Epac1-camps, was used to monitor the time course of cytosolic cAMP changes. A pulse application of the β-adrenoceptor (β-AR) agonist isoproterenol (Iso) induced a transient FRET signal. Both β1- and β2-AR antagonists decreased the signal amplitude without affecting its kinetics. The non-selective PDE inhibitor (IBMX) dramatically increased the amplitude and delayed the recovery phase of Iso response, in agreement with a role of PDEs in degrading cAMP produced by Iso. Whereas PDE1, PDE3 and PDE7 blockades [with MIMX, cilostamide (Cil) and BRL 50481 (BRL), respectively] had no or minor effect on Iso response, PDE4 inhibition [with Ro-20-1724 (Ro)] strongly increased its amplitude and delayed its recovery. When Ro was applied concomitantly with MIMX or Cil (but not with BRL), the Iso response was drastically further prolonged. PDE4 inhibition similarly prolonged both β1- and β2-AR-mediated responses. When a membrane-targeted FRET sensor was used, PDE3 and PDE4 acted in a synergistic manner to hydrolyze the submembrane cAMP produced either at baseline or after β-AR stimulation.Conclusion/Significance
Our study underlines the importance of cAMP-PDEs in the dynamic control of intracellular cAMP signals in RASMCs, and demonstrates the prominent role of PDE4 in limiting β-AR responses. PDE4 inhibition unmasks an effect of PDE1 and PDE3 on cytosolic cAMP hydrolyzis, and acts synergistically with PDE3 inhibition at the submembrane compartment. This suggests that mixed PDE4/PDE1 or PDE4/PDE3 inhibitors would be attractive to potentiate cAMP-related functions in vascular cells. 相似文献10.
Dynamic Light-Scattering Evidence for the Flexibility of Native Muscle Thin Filaments 总被引:1,自引:1,他引:0
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We have obtained clear evidence for the flexibility of native scallop adductor thin filaments by studying the temperature and ionic strength dependence of the average decay constants obtained from intensity fluctuation spectroscopic (IFS) measurements. The low-angle (10-25°), average decay constants obtained from time autocorrelation functions of scattered light were independent of concentration (0.08-1.3 mg/ml), scaled with the ratio of temperature to solvent viscosity, T/η, over a range of 4-45°C, and yielded a value for the translational diffusion coefficient of DT5°C = (1.24 ± 0.06) × 10-8 cm2/s. From this value and the Broersma relation for rigid rods, we find an average filament length of 1.06 ± 0.06 μm. Quantitative sodium dodecyl sulfate polyacrylamide gel electrophoresis showed that at high temperatures (> 35°C) or in 0.6 M NaCl, tropomyosin completely dissociates from native thin filaments. Decay constants from high-angle (60-150°C) IFS temperature dependence measurements do not scale with T/η and hence do not show the temperature dependence expected for rigid rods. The differences are not due to any change in length distribution of filaments with temperature or to the free tropomyosin in solution, but are attributed to nonrigid motions of the filaments. Similar experiments on samples in high- and low-salt solvents gave results consistent with this interpretation. 相似文献
11.
Alexandre Beautrait Kevin R. Michalski Thomas S. Lopez Katelynn M. Mannix Devin J. McDonald Amber R. Cutter Christopher B. Medina Aaron M. Hebert Charnelle J. Francis Michel Bouvier John J. G. Tesmer Rachel Sterne-Marr 《The Journal of biological chemistry》2014,289(36):25262-25275
G protein-coupled receptor kinases (GRKs) phosphorylate agonist-occupied receptors initiating the processes of desensitization and β-arrestin-dependent signaling. Interaction of GRKs with activated receptors serves to stimulate their kinase activity. The extreme N-terminal helix (αN), the kinase small lobe, and the active site tether (AST) of the AGC kinase domain have previously been implicated in mediating the allosteric activation. Expanded mutagenesis of the αN and AST allowed us to further assess the role of these two regions in kinase activation and receptor phosphorylation in vitro and in intact cells. We also developed a bioluminescence resonance energy transfer-based assay to monitor the recruitment of GRK2 to activated α2A-adrenergic receptors (α2AARs) in living cells. The bioluminescence resonance energy transfer signal exhibited a biphasic response to norepinephrine concentration, suggesting that GRK2 is recruited to Gβγ and α2AAR with EC50 values of 15 nm and 8 μm, respectively. We show that mutations in αN (L4A, V7E, L8E, V11A, S12A, Y13A, and M17A) and AST (G475I, V477D, and I485A) regions impair or potentiate receptor phosphorylation and/or recruitment. We suggest that a surface of GRK2, including Leu4, Val7, Leu8, Val11, and Ser12, directly interacts with receptors, whereas residues such as Asp10, Tyr13, Ala16, Met17, Gly475, Val477, and Ile485 are more important for kinase domain closure and activation. Taken together with data on GRK1 and GRK6, our data suggest that all three GRK subfamilies make conserved interactions with G protein-coupled receptors, but there may be unique interactions that influence selectivity. 相似文献
12.
Diogo E.P. Pinto Gonca Erdemci-Tandogan M. Lisa Manning Nuno A.M. Araújo 《Biophysical journal》2020,119(11):2299
The structure and dynamics of tissue cultures depend strongly on the physical and chemical properties of the underlying substrate. Inspired by previous advances in the context of inorganic materials, the use of patterned culture surfaces has been proposed as an effective way to induce space-dependent properties in cell tissues. However, cells move and diffuse, and the transduction of external stimuli to biological signals is not instantaneous. Here, we show that the fidelity of patterns to demix tissue cells depends on the relation between the diffusion (τD) and adaptation (τ) times. Numerical results for the self-propelled Voronoi model reveal that the fidelity decreases with τ/τD, a result that is reproduced by a continuum reaction-diffusion model. Based on recent experimental results for single cells, we derive a minimal length scale for the patterns in the substrate that depends on τ/τD and can be much larger than the cell size. 相似文献
13.
Jergic S Ozawa K Williams NK Su XC Scott DD Hamdan SM Crowther JA Otting G Dixon NE 《Nucleic acids research》2007,35(9):2813-2824
The τ subunit of Escherichia coli DNA polymerase III holoenzyme interacts with the α subunit through its C-terminal Domain V, τC16. We show that the extreme C-terminal region of τC16 constitutes the site of interaction with α. The τC16 domain, but not a derivative of it with a C-terminal deletion of seven residues (τC16Δ7), forms an isolable complex with α. Surface plasmon resonance measurements were used to determine the dissociation constant (KD) of the α−τC16 complex to be ~260pM. Competition with immobilized τC16 by τC16 derivatives for binding to α gave values of KD of 7μM for the α−τC16Δ7 complex. Low-level expression of the genes encoding τC16 and τC167, but not τC16Δ11, is lethal to E. coli. Suppression of this lethal phenotype enabled selection of mutations in the 3′ end of the τC16 gene, that led to defects in α binding. The data suggest that the unstructured C-terminus of τ becomes folded into a helix–loop–helix in its complex with α. An N-terminally extended construct, τC24, was found to bind DNA in a salt-sensitive manner while no binding was observed for τC16, suggesting that the processivity switch of the replisome functionally involves Domain IV of τ. 相似文献
14.
Ga?l Manes Pallavi Cheguru Anurima Majumder Béatrice Bocquet Audrey Sénéchal Nikolai O. Artemyev Christian P. Hamel Philippe Brabet 《PloS one》2014,9(4)
Autosomal dominant congenital stationary night blindness (adCSNB) is caused by mutations in three genes of the rod phototransduction cascade, rhodopsin (RHO), transducin α-subunit (GNAT1), and cGMP phosphodiesterase type 6 β-subunit (PDE6B). In most cases, the constitutive activation of the phototransduction cascade is a prerequisite to cause adCSNB. The unique adCSNB-associated PDE6B mutation found in the Rambusch pedigree, the substitution p.His258Asn, leads to rod photoreceptors desensitization. Here, we report a three-generation French family with adCSNB harboring a novel PDE6B mutation, the duplication, c.928-9_940dup resulting in a tyrosine to cysteine substitution at codon 314, a frameshift, and a premature termination (p.Tyr314Cysfs*50). To understand the mechanism of the PDE6β1-314fs*50 mutant, we examined the properties of its PDE6-specific portion, PDE6β1-313. We found that PDE6β1-313 maintains the ability to bind noncatalytic cGMP and the inhibitory γ-subunit (Pγ), and interferes with the inhibition of normal PDE6αβ catalytic subunits by Pγ. Moreover, both truncated forms of the PDE6β protein, PDE6β1-313 and PDE6β1-314fs*50 expressed in rods of transgenic X. laevis are targeted to the phototransduction compartment. We hypothesize that in affected family members the p.Tyr314Cysfs*50 change results in the production of the truncated protein, which binds Pγ and causes constitutive activation of the phototransduction thus leading to the absence of rod adaptation. 相似文献
15.
Haruhisa Okawa Johan Pahlberg Fred Rieke Lutz Birnbaumer Alapakkam P. Sampath 《The Journal of general physiology》2010,136(4):443-454
The high sensitivity of scotopic vision depends on the efficient retinal processing of single photon responses generated by individual rod photoreceptors. At the first synapse in the mammalian retina, rod outputs are pooled by a rod “ON” bipolar cell, which uses a G-protein signaling cascade to enhance the fidelity of the single photon response under conditions where few rods absorb light. Here we show in mouse rod bipolar cells that both splice variants of the Go α subunit, Gαo1 and Gαo2, mediate light responses under the control of mGluR6 receptors, and their coordinated action is critical for maximizing sensitivity. We found that the light response of rod bipolar cells was primarily mediated by Gαo1, but the loss of Gαo2 caused a reduction in the light sensitivity. This reduced sensitivity was not attributable to the reduction in the total number of Go α subunits, or the altered balance of expression levels between the two splice variants. These results indicate that Gαo1 and Gαo2 both mediate a depolarizing light response in rod bipolar cells without occluding each other’s actions, suggesting they might act independently on a common effector. Thus, Gαo2 plays a role in improving the sensitivity of rod bipolar cells through its action with Gαo1. The coordinated action of two splice variants of a single Gα may represent a novel mechanism for the fine control of G-protein activity. 相似文献
16.
Mathematical analysis of the dependence of cell potential on external potassium in corn roots 总被引:10,自引:5,他引:5
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The K+ dependence of normal (ψ) and diffusion (ψD) potentials in corn roots [Zea mays L., hybrid (A619 × Oh43) × A632] was determined experimentally and analyzed with respect to the parameter ξ [defined as exp (F ψ/RT)]. In the presence of 10 micromolar carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP), ψ behaved as expected of a diffusion potential. Based upon the assumptions (a) that FCCP did not change any term of the Goldman-Hodgkin-Katz equation, and (b) that total potential was functionally the algebraic sum of ψD and ψP (the deviation from ψD due to an electrogenic system), ψP was found to be a complex function of external potassium and to have a minimum value of 0.69 millimolar K ion activity outside the cell. Analysis of ψ allowed us to develop an equation which predicts a complicated K+ dependence of ψ such as that found by Mertz and Higinbotham (Membrane Transport in Plants and Plant Organelles. Springer-Verlag 1974). 相似文献
17.
Nucleotide polymorphism at 12 nuclear loci was studied in Scots pine populations across an environmental gradient in Scotland, to evaluate the impacts of demographic history and selection on genetic diversity. At eight loci, diversity patterns were compared between Scottish and continental European populations. At these loci, a similar level of diversity (θsil=∼0.01) was found in Scottish vs mainland European populations, contrary to expectations for recent colonization, however, less rapid decay of linkage disequilibrium was observed in the former (ρ=0.0086±0.0009, ρ=0.0245±0.0022, respectively). Scottish populations also showed a deficit of rare nucleotide variants (multi-locus Tajima''s D=0.316 vs D=−0.379) and differed significantly from mainland populations in allelic frequency and/or haplotype structure at several loci. Within Scotland, western populations showed slightly reduced nucleotide diversity (πtot=0.0068) compared with those from the south and east (0.0079 and 0.0083, respectively) and about three times higher recombination to diversity ratio (ρ/θ=0.71 vs 0.15 and 0.18, respectively). By comparison with results from coalescent simulations, the observed allelic frequency spectrum in the western populations was compatible with a relatively recent bottleneck (0.00175 × 4Ne generations) that reduced the population to about 2% of the present size. However, heterogeneity in the allelic frequency distribution among geographical regions in Scotland suggests that subsequent admixture of populations with different demographic histories may also have played a role. 相似文献
18.
On the Origin and the Signal-Shaping Mechanism of the Fast Photosignal in the Vertebrate Retina 总被引:1,自引:0,他引:1
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Fast photosignals (FPS) with R1 and R2 components were measured in retinas of cattle, rat, and frog within a temperature range of 0° to 60°C. Except for temperatures near 0°C the signal rise of the R1 component was determined by the duration of the exciting flash. The kinetics of the R2 component and the meta transition of rhodopsin in the cattle and rat retina were compared. For the analysis of the FPS it is presupposed that the signal is produced by light-induced charges on the outer segment envelope membrane that spread onto the whole plasma membrane of the photoreceptor cell. To a good approximation, this mechanism can be described by a model circuit with two distinct capacitors. In this model, the charging capacitance of the pigmented outer segment envelope membrane and the capacitance of the receptor's nonpigmented plasma membrane are connected via the extra- and intracellular electrolyte resistances. The active charging is explained by two independent processes, both with exponential rise (R1 and R2), that are due to charge displacements within the pigmented envelope membrane. The time constant τ2 of the R2 membrane charging process shows a strong temperature dependence that of the charge redistribution, τr, a weak one. In frog and cattle retinas the active charging is much slower within a large temperature range than the passive charge redistribution. From the two-capacitor model it follows for τr « τ2 that the rise of the R2 component is determined by τr, whereas the decay is given by τ2. For the rat retina, however, τ2 approaches τr at physiological temperatures and becomes <τr above 45°C. In this temperature range where τ2 ≈ τr, both processes affect rise and decay of the photosignal. The absolute values of τr are in good accordance with the known electric parameters of the photoreceptors. At least in the cattle retina, the time constant τ2 is identical with that of the slow component of the meta II formation. The strong temperature dependence of the meta transition time gives rise to the marked decrease of the R2 amplitude with falling temperature. As the R1 rise could not be fully time resolved the signal analysis does not yield the time constant τ1 of the R1 generating process. It could be established, however, within the whole temperature range that the decay of the R1 component is determined by τr. Using an extended model that allows for membrane leakage, we show that in normal ringer solution the membrane time constant does not influence the signal time-course and amplitude. 相似文献
19.
Gemma Navarro Estefania Moreno Jordi Bonaventura Marc Brugarolas Daniel Farré David Aguinaga Josefa Mallol Antoni Cortés Vicent Casadó Carmen Lluís Sergi Ferre Rafael Franco Enric Canela Peter J. McCormick 《PloS one》2013,8(4)
Under normal conditions the brain maintains a delicate balance between inputs of reward seeking controlled by neurons containing the D1-like family of dopamine receptors and inputs of aversion coming from neurons containing the D2-like family of dopamine receptors. Cocaine is able to subvert these balanced inputs by altering the cell signaling of these two pathways such that D1 reward seeking pathway dominates. Here, we provide an explanation at the cellular and biochemical level how cocaine may achieve this. Exploring the effect of cocaine on dopamine D2 receptors function, we present evidence of σ1 receptor molecular and functional interaction with dopamine D2 receptors. Using biophysical, biochemical, and cell biology approaches, we discovered that D2 receptors (the long isoform of the D2 receptor) can complex with σ1 receptors, a result that is specific to D2 receptors, as D3 and D4 receptors did not form heteromers. We demonstrate that the σ1-D2 receptor heteromers consist of higher order oligomers, are found in mouse striatum and that cocaine, by binding to σ1 -D2 receptor heteromers, inhibits downstream signaling in both cultured cells and in mouse striatum. In contrast, in striatum from σ1 knockout animals these complexes are not found and this inhibition is not seen. Taken together, these data illuminate the mechanism by which the initial exposure to cocaine can inhibit signaling via D2 receptor containing neurons, destabilizing the delicate signaling balance influencing drug seeking that emanates from the D1 and D2 receptor containing neurons in the brain. 相似文献
20.
The mechanism of response of two potential-sensitive dyes, diOC2(5) (3,3′-diethyloxadicarbocyanine iodide) and oxonol V (bis-[3-phenyl-5-oxoisoxazol-4-yl]pentamethine oxonol), were studied by using steady-state and time-resolved fluorescence techniques. The lipid concentration dependence of the Δψ (membrane potential)-induced change in total fluorescence intensity was quite different for these two dyes. Time-resolved fluorescence measurements showed that the fluorescence decay of these dyes in membranes could be resolved into at least three exponentials. Δψ-induced changes in the levels of these three populations were also measured under a variety of conditions. In the case of diOC2(5) an inside negative Δψ increased the levels of the bound forms. This shows that diOC2(5) responds to Δψ mainly by an “on-off” mechanism whereby Δψ perturbs the membrane-water partition coefficient of the dye. The Δψ-induced changes approached zero when the dye was totally membrane bound. In contrast, the Δψ-induced response of oxonol V increased with increased membrane binding. An inside negative Δψ decreased the level of the bound form with a longer lifetime. This shows that the mechanism of response of oxonol V is a Δψ-induced shift in the equilibrium between bound forms of the dye. 相似文献