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1.
Over the past few years, it has become clear that an important mechanism by which large-conductance Ca2+-activated K+ channel (BKCa) activity is regulated is the tissue-specific expression of auxiliary β subunits. The first of these to be identified, β1, is expressed predominately in smooth muscle and causes dramatic effects, increasing the apparent affinity of the channel for Ca2+ 10-fold at 0 mV, and shifting the range of voltages over which the channel activates −80 mV at 9.1 μM Ca2+. With this study, we address the question: which aspects of BKCa gating are altered by β1 to bring about these effects: Ca2+ binding, voltage sensing, or the intrinsic energetics of channel opening? The approach we have taken is to express the β1 subunit together with the BKCa α subunit in Xenopus oocytes, and then to compare β1''s steady state effects over a wide range of Ca2+ concentrations and membrane voltages to those predicted by allosteric models whose parameters have been altered to mimic changes in the aspects of gating listed above. The results of our analysis suggest that much of β1''s steady state effects can be accounted for by a reduction in the intrinsic energy the channel must overcome to open and a decrease in its voltage sensitivity, with little change in the affinity of the channel for Ca2+ when it is either open or closed. Interestingly, however, the small changes in Ca2+ binding affinity suggested by our analysis (Kc 7.4 μM → 9.6 μM; Ko = 0.80 μM → 0.65 μM) do appear to be functionally important. We also show that β1 affects the mSlo conductance–voltage relation in the essential absence of Ca2+, shifting it +20 mV and reducing its apparent gating charge 38%, and we develop methods for distinguishing between alterations in Ca2+ binding and other aspects of BKCa channel gating that may be of general use. 相似文献
2.
Lahti M Bligt E Niskanen H Parkash V Brandt AM Jokinen J Patrikainen P Käpylä J Heino J Salminen TA 《The Journal of biological chemistry》2011,286(50):43343-43351
We have analyzed the structure and function of the integrin α1I domain harboring a gain-of-function mutation E317A. To promote protein crystallization, a double variant with an additional C139S mutation was used. In cell adhesion assays, the E317A mutation promoted binding to collagen. Similarly, the double mutation C139S/E317A increased adhesion compared with C139S alone. Furthermore, soluble α1I C139S/E317A was a higher avidity collagen binder than α1I C139S, indicating that the double variant represents an activated form. The crystal structure of the activated variant of α1I was solved at 1.9 Å resolution. The E317A mutation results in the unwinding of the αC helix, but the metal ion has moved toward loop 1, instead of loop 2 in the open α2I. Furthermore, unlike in the closed αI domains, the metal ion is pentacoordinated and, thus, prepared for ligand binding. Helix 7, which has moved downward in the open α2I structure, has not changed its position in the activated α1I variant. During the integrin activation, Glu335 on helix 7 binds to the metal ion at the metal ion-dependent adhesion site (MIDAS) of the β1 subunit. Interestingly, in our cell adhesion assays E317A could activate collagen binding even after mutating Glu335. This indicates that the stabilization of helix 7 into its downward position is not required if the α1 MIDAS is already open. To conclude, the activated α1I domain represents a novel conformation of the αI domain, mimicking the structural state where the Arg287-Glu317 ion pair has just broken during the integrin activation. 相似文献
3.
Yusheng Jiang Yun Huang Hing-Cheung Wong Yubin Zhou Xue Wang Jun Yang Randy A. Hall Edward M. Brown Jenny J. Yang 《The Journal of biological chemistry》2010,285(43):33463-33474
Metabotropic glutamate receptor 1α (mGluR1α) exerts important effects on numerous neurological processes. Although mGluR1α is known to respond to extracellular Ca2+ ([Ca2+]o) and the crystal structures of the extracellular domains (ECDs) of several mGluRs have been determined, the calcium-binding site(s) and structural determinants of Ca2+-modulated signaling in the Glu receptor family remain elusive. Here, we identify a novel Ca2+-binding site in the mGluR1α ECD using a recently developed computational algorithm. This predicted site (comprising Asp-318, Glu-325, and Asp-322 and the carboxylate side chain of the receptor agonist, Glu) is situated in the hinge region in the ECD of mGluR1α adjacent to the reported Glu-binding site, with Asp-318 involved in both Glu and calcium binding. Mutagenesis studies indicated that binding of Glu and Ca2+ to their distinct but partially overlapping binding sites synergistically modulated mGluR1α activation of intracellular Ca2+ ([Ca2+]i) signaling. Mutating the Glu-binding site completely abolished Glu signaling while leaving its Ca2+-sensing capability largely intact. Mutating the predicted Ca2+-binding residues abolished or significantly reduced the sensitivity of mGluR1α not only to [Ca2+]o and [Gd3+]o but also, in some cases, to Glu. The dual activation of mGluR1α by [Ca2+]o and Glu has important implications for the activation of other mGluR subtypes and related receptors. It also opens up new avenues for developing allosteric modulators of mGluR function that target specific human diseases. 相似文献
4.
Yan Yang Xin Wu Peichun Gui Jianbo Wu Jian-Zhong Sheng Shizhang Ling Andrew P. Braun George E. Davis Michael J. Davis 《The Journal of biological chemistry》2010,285(1):131-141
Large conductance, calcium-activated K+ (BK) channels are important regulators of cell excitability and recognized targets of intracellular kinases. BK channel modulation by tyrosine kinases, including focal adhesion kinase and c-src, suggests their potential involvement in integrin signaling. Recently, we found that fibronectin, an endogenous α5β1 integrin ligand, enhances BK channel current through both Ca2+- and phosphorylation-dependent mechanisms in vascular smooth muscle. Here, we show that macroscopic currents from HEK 293 cells expressing murine BK channel α-subunits (mSlo) are acutely potentiated following α5β1 integrin activation. The effect occurs in a Ca2+-dependent manner, 1–3 min after integrin engagement. After integrin activation, normalized conductance-voltage relations for mSlo are left-shifted at free Ca2+ concentrations ≥1 μm. Overexpression of human c-src with mSlo, in the absence of integrin activation, leads to similar shifts in mSlo Ca2+ sensitivity, whereas overexpression of catalytically inactive c-src blocks integrin-induced potentiation. However, neither integrin activation nor c-src overexpression potentiates current in BK channels containing a point mutation at Tyr-766. Biochemical tests confirmed the critical importance of residue Tyr-766 in integrin-induced channel phosphorylation. Thus, BK channel activity is enhanced by α5β1 integrin activation, likely through an intracellular signaling pathway involving c-src phosphorylation of the channel α-subunit at Tyr-766. The net result is increased current amplitude, enhanced Ca2+ sensitivity, and rate of activation of the BK channel, which would collectively promote smooth muscle hyperpolarization in response to integrin-extracellular matrix interactions. 相似文献
5.
Paul H. Weinreb Sheng Li Sharon X. Gao Tong Liu R. Blake Pepinsky Justin A. Caravella Jun H. Lee Virgil L. Woods Jr. 《The Journal of biological chemistry》2012,287(39):32897-32912
We have applied hydrogen-deuterium exchange mass spectrometry, in conjunction with differential scanning calorimetry and protein stability analysis, to examine solution dynamics of the integrin α1 I domain induced by the binding of divalent cations, full-length type IV collagen, or a function-blocking monoclonal antibody. These studies revealed features of integrin activation and α1I-ligand complexes that were not detected by static crystallographic data. Mg2+ and Mn2+ stabilized α1I but differed in their effects on exchange rates in the αC helix. Ca2+ impacted α1I conformational dynamics without altering its gross thermal stability. Interaction with collagen affected the exchange rates in just one of three metal ion-dependent adhesion site (MIDAS) loops, suggesting that MIDAS loop 2 plays a primary role in mediating ligand binding. Collagen also induced changes consistent with increased unfolding in both the αC and allosteric C-terminal helices of α1I. The antibody AQC2, which binds to α1I in a ligand-mimetic manner, also reduced exchange in MIDAS loop 2 and increased exchange in αC, but it did not impact the C-terminal region. This is the first study to directly demonstrate the conformational changes induced upon binding of an integrin I domain to a full-length collagen ligand, and it demonstrates the utility of the deuterium exchange mass spectrometry method to study the solution dynamics of integrin/ligand and integrin/metal ion interactions. Based on the ligand and metal ion binding data, we propose a model for collagen-binding integrin activation that explains the differing abilities of Mg2+, Mn2+, and Ca2+ to activate I domain-containing integrins. 相似文献
6.
Jason Y. Jiang Mulpuri Nagaraju Rebecca C. Meyer Li Zhang Donald Hamelberg Randy A. Hall Edward M. Brown P. Jeffrey Conn Jenny J. Yang 《The Journal of biological chemistry》2014,289(3):1649-1661
Metabotropic glutamate receptor 1α (mGluR1α), a member of the family C G protein-coupled receptors, is emerging as a potential drug target for various disorders, including chronic neuronal degenerative diseases. In addition to being activated by glutamate, mGluR1α is also modulated by extracellular Ca2+. However, the underlying mechanism is unknown. Moreover, it has long been challenging to develop receptor-specific agonists due to homologies within the mGluR family, and the Ca2+-binding site(s) on mGluR1α may provide an opportunity for receptor-selective targeting by therapeutics. In the present study, we show that our previously predicted Ca2+-binding site in the hinge region of mGluR1α is adjacent to the site where orthosteric agonists and antagonists bind on the extracellular domain of the receptor. Moreover, we found that extracellular Ca2+ enhanced mGluR1α-mediated intracellular Ca2+ responses evoked by the orthosteric agonist l-quisqualate. Conversely, extracellular Ca2+ diminished the inhibitory effect of the mGluR1α orthosteric antagonist (S)-α-methyl-4-carboxyphenylglycine. In addition, selective positive (Ro 67-4853) and negative (7-(hydroxyimino)cyclopropa[b]chromen-1a-carboxylate ethyl ester) allosteric modulators of mGluR1α potentiated and inhibited responses to extracellular Ca2+, respectively, in a manner similar to their effects on the response of mGluR1α to glutamate. Mutations at residues predicted to be involved in Ca2+ binding, including E325I, had significant effects on the modulation of responses to the orthosteric agonist l-quisqualate and the allosteric modulator Ro 67-4853 by extracellular Ca2+. These studies reveal that binding of extracellular Ca2+ to the predicted Ca2+-binding site in the extracellular domain of mGluR1α modulates not only glutamate-evoked signaling but also the actions of both orthosteric ligands and allosteric modulators on mGluR1α. 相似文献
7.
Daniele Catalucci Deng-Hong Zhang Jaime DeSantiago Franck Aimond Guillaume Barbara Jean Chemin Désiré Bonci Eckard Picht Francesca Rusconi Nancy D. Dalton Kirk L. Peterson Sylvain Richard Donald M. Bers Joan Heller Brown Gianluigi Condorelli 《The Journal of cell biology》2009,184(6):923-933
The insulin IGF-1–PI3K–Akt signaling pathway has been suggested to
improve cardiac inotropism and increase Ca2+ handling through
the effects of the protein kinase Akt. However, the underlying molecular
mechanisms remain largely unknown. In this study, we provide evidence for an
unanticipated regulatory function of Akt controlling L-type Ca2+
channel (LTCC) protein density. The pore-forming channel subunit
Cavα1 contains highly conserved PEST sequences (signals for
rapid protein degradation), and in-frame deletion of these PEST sequences
results in increased Cavα1 protein levels. Our findings show
that Akt-dependent phosphorylation of Cavβ2, the LTCC chaperone
for Cavα1, antagonizes Cavα1 protein
degradation by preventing Cavα1 PEST sequence recognition,
leading to increased LTCC density and the consequent modulation of
Ca2+ channel function. This novel mechanism by which Akt
modulates LTCC stability could profoundly influence cardiac myocyte
Ca2+ entry, Ca2+ handling, and
contractility. 相似文献
8.
Carefully soaking crystals with Arg-Gly-Asp (RGD) peptides, we captured eight distinct RGD-bound conformations of the αIIbβ3 integrin headpiece. Starting from the closed βI domain conformation, we saw six intermediate βI conformations and finally the fully open βI with the hybrid domain swung out in the crystal lattice. The β1-α1 backbone that hydrogen bonds to the Asp side chain of RGD was the first element to move followed by adjacent to metal ion-dependent adhesion site Ca2+, α1 helix, α1’ helix, β6-α7 loop, α7 helix, and hybrid domain. We define in atomic detail how conformational change was transmitted over long distances in integrins, 40 Å from the ligand binding site to the opposite end of the βI domain and 80 Å to the far end of the hybrid domain. During these movements, RGD slid in its binding groove toward αIIb, and its Arg side chain became ordered. RGD concentration requirements in soaking suggested a >200-fold higher affinity after opening. The thermodynamic cycle shows how higher affinity pays the energetic cost of opening. 相似文献
9.
Albert Gubern Miquel Barcel��-Torns David Barneda Jos�� M. L��pez Roser Masgrau Fernando Picatoste Charles E. Chalfant Jes��s Balsinde Mar��a A. Balboa Enrique Claro 《The Journal of biological chemistry》2009,284(47):32359-32369
The biogenesis of lipid droplets (LD) induced by serum depends on group IVA phospholipase A2 (cPLA2α). This work dissects the pathway leading to cPLA2α activation and LD biogenesis. Both processes were Ca2+-independent, as they took place after pharmacological blockade of Ca2+ transients elicited by serum or chelation with 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid tetrakis(acetoxymethyl ester). The single mutation D43N in cPLA2α, which abrogates its Ca2+ binding capacity and translocation to membranes, did not affect enzyme activation and formation of LD. In contrast, the mutation S505A did not affect membrane relocation of the enzyme in response to Ca2+ but prevented its phosphorylation, activation, and the appearance of LD. Expression of specific activators of different mitogen-activated protein kinases showed that phosphorylation of cPLA2α at Ser-505 is due to JNK. This was confirmed by pharmacological inhibition and expression of a dominant-negative form of the upstream activator MEKK1. LD biogenesis was accompanied by increased synthesis of ceramide 1-phosphate. Overexpression of its synthesizing enzyme ceramide kinase increased phosphorylation of cPLA2α at Ser-505 and formation of LD, and its down-regulation blocked the phosphorylation of cPLA2α and LD biogenesis. These results demonstrate that LD biogenesis induced by serum is regulated by JNK and ceramide kinase. 相似文献
10.
Fang Kong Andrés J. García A. Paul Mould Martin J. Humphries Cheng Zhu 《The Journal of cell biology》2009,185(7):1275-1284
Binding of integrins to ligands provides anchorage and signals for the cell, making them prime candidates for mechanosensing molecules. How force regulates integrin–ligand dissociation is unclear. We used atomic force microscopy to measure the force-dependent lifetimes of single bonds between a fibronectin fragment and an integrin α5β1-Fc fusion protein or membrane α5β1. Force prolonged bond lifetimes in the 10–30-pN range, a counterintuitive behavior called catch bonds. Changing cations from Ca2+/Mg2+ to Mg2+/EGTA and to Mn2+ caused longer lifetime in the same 10–30-pN catch bond region. A truncated α5β1 construct containing the headpiece but not the legs formed longer-lived catch bonds that were not affected by cation changes at forces <30 pN. Binding of monoclonal antibodies that induce the active conformation of the integrin headpiece shifted catch bonds to a lower force range. Thus, catch bond formation appears to involve force-assisted activation of the headpiece but not integrin extension. 相似文献
11.
M.S. Chen E.A.C. Almeida A.-P.J. Huovila Y. Takahashi L.M. Shaw A.M. Mercurio J.M. White 《The Journal of cell biology》1999,144(3):549-561
Integrins can exist in different functional states with low or high binding capacity for particular ligands. We previously provided evidence that the integrin α6β1, on mouse eggs and on α6-transfected cells, interacted with the disintegrin domain of the sperm surface protein ADAM 2 (fertilin β). In the present study we tested the hypothesis that different states of α6β1 interact with fertilin and laminin, an extracellular matrix ligand for α6β1. Using α6-transfected cells we found that treatments (e.g., with phorbol myristate acetate or MnCl2) that increased adhesion to laminin inhibited sperm binding. Conversely, treatments that inhibited laminin adhesion increased sperm binding. Next, we compared the ability of fluorescent beads coated with either fertilin β or with the laminin E8 fragment to bind to eggs. In Ca2+-containing media, fertilin β beads bound to eggs via an interaction mediated by the disintegrin loop of fertilin β and by the α6 integrin subunit. In Ca2+-containing media, laminin E8 beads did not bind to eggs. Treatment of eggs with phorbol myristate acetate or with the actin disrupting agent, latrunculin A, inhibited fertilin bead binding, but did not induce laminin E8 bead binding. Treatment of eggs with Mn2+ dramatically increased laminin E8 bead binding, and inhibited fertilin bead binding. Our results provide the first evidence that different states of an integrin (α6β1) can interact with an extracellular matrix ligand (laminin) or a membrane-anchored cell surface ligand (ADAM 2). 相似文献
12.
Omar Ramadan Yongxia Qu Raj Wadgaonkar Ghayath Baroudi Eddy Karnabi Mohamed Chahine Mohamed Boutjdir 《The Journal of biological chemistry》2009,284(8):5042-5049
The novel α1D L-type Ca2+ channel is expressed
in supraventricular tissue and has been implicated in the pacemaker activity
of the heart and in atrial fibrillation. We recently demonstrated that PKA
activation led to increased α1D Ca2+ channel
activity in tsA201 cells by phosphorylation of the channel protein. Here we
sought to identify the phosphorylated PKA consensus sites on the
α1 subunit of the α1D Ca2+
channel by generating GST fusion proteins of the intracellular loops, N
terminus, proximal and distal C termini of the α1 subunit of
α1D Ca2+ channel. An in vitro PKA kinase
assay was performed for the GST fusion proteins, and their phosphorylation was
assessed by Western blotting using either anti-PKA substrate or
anti-phosphoserine antibodies. Western blotting showed that the N terminus and
C terminus were phosphorylated. Serines 1743 and 1816, two PKA consensus
sites, were phosphorylated by PKA and identified by mass spectrometry. Site
directed mutagenesis and patch clamp studies revealed that serines 1743 and
1816 were major functional PKA consensus sites. Altogether, biochemical and
functional data revealed that serines 1743 and 1816 are major functional PKA
consensus sites on the α1 subunit of α1D
Ca2+ channel. These novel findings provide new insights into the
autonomic regulation of the α1D Ca2+ channel in
the heart.L-type Ca2+ channels are essential for the generation of normal
cardiac rhythm, for induction of rhythm propagation through the
atrioventricular node and for the contraction of the atrial and ventricular
muscles
(1–5).
L-type Ca2+ channel is a multisubunit complex including
α1, β and α2/δ subunits
(5–7).
The α1 subunit contains the voltage sensor, the selectivity
filter, the ion conduction pore, and the binding sites for all known
Ca2+ channel blockers
(6–9).
While α1C Ca2+ channel is expressed in the atria
and ventricles of the heart
(10–13),
expression of α1D Ca2+ channel is restricted to
the sinoatrial (SA)2
and atrioventricular (AV) nodes, as well as in the atria, but not in the adult
ventricles (2,
3,
10).Only recently it has been realized that α1D along with
α1C Ca2+ channels contribute to L-type
Ca2+ current (ICa-L) and they both play important but
unique roles in the physiology/pathophysiology of the heart
(6–9).
Compared with α1C, α1D L-type
Ca2+ channel activates at a more negative voltage range and shows
slower current inactivation during depolarization
(14,
15). These properties may
allow α1D Ca2+ channel to play critical roles in
SA and AV nodes function. Indeed, α1D Ca2+ channel
knock-out mice exhibit significant SA dysfunction and various degrees of AV
block (12,
16–19).The modulation of α1C Ca2+ channel by
cAMP-dependent PKA phosphorylation has been extensively studied, and the C
terminus of α1 was identified as the site of the modulation
(20–22).
Our group was the first to report that 8-bromo-cAMP (8-Br-cAMP), a
membrane-permeable cAMP analog, increased α1D Ca2+
channel activity using patch clamp studies
(2). However, very little is
known about potential PKA phosphorylation consensus motifs on the
α1D Ca2+ channel. We therefore hypothesized that
the C terminus of the α1 subunit of the α1D
Ca2+ channel mediates its modulation by cAMP-dependent PKA
pathway. 相似文献
13.
Merav Marom Yamit Hagalili Ariel Sebag Lior Tzvier Daphne Atlas 《The Journal of biological chemistry》2010,285(10):6996-7005
The role of the L-type calcium channel (Cav1.2) as a molecular switch that triggers secretion prior to Ca2+ transport has previously been demonstrated in bovine chromaffin cells and rat pancreatic beta cells. Here, we examined the effect of specific Cav1.2 allosteric modulators, BayK 8644 (BayK) and FPL64176 (FPL), on the kinetics of catecholamine release, as monitored by amperometry in single bovine chromaffin cells. We show that 2 μm BayK or 0.5 μm FPL accelerates the rate of catecholamine secretion to a similar extent in the presence either of the permeable Ca2+ and Ba2+ or the impermeable charge carrier La3+. These results suggest that structural rearrangements generated through the binding of BayK or FPL, by altering the channel activity, could affect depolarization-evoked secretion prior to cation transport. FPL also accelerated the rate of secretion mediated by a Ca2+-impermeable channel made by replacing the wild type α11.2 subunit was replaced with the mutant α11.2/L775P. Furthermore, BayK and FPL modified the kinetic parameters of the fusion pore formation, which represent the initial contact between the vesicle lumen and the extracellular medium. A direct link between the channel activity and evoked secretion lends additional support to the view that the voltage-gated Ca2+ channels act as a signaling molecular switch, triggering secretion upstream to ion transport into the cell. 相似文献
14.
15.
Aguang Dai Feng Ye Dianne W. Taylor Guiqing Hu Mark H. Ginsberg Kenneth A. Taylor 《The Journal of biological chemistry》2015,290(45):27168-27175
Increased ligand binding to integrin (“activation”) underpins many biological processes, such as leukocyte trafficking, cell migration, host-pathogen interaction, and hemostasis. Integrins exist in several conformations, ranging from compact and bent to extended and open. However, the exact conformation of membrane-embedded, full-length integrin bound to its physiological macromolecular ligand is still unclear. Integrin αIIbβ3, the most abundant integrin in platelets, has been a prototype for integrin activation studies. Using negative stain electron microscopy and nanodisc-embedding to provide a membrane-like environment, we visualized the conformation of full-length αIIbβ3 in both a Mn2+-activated, ligand-free state and a Mn2+-activated, fibrin-bound state. Activated but ligand-free integrins exist mainly in the compact conformation, whereas fibrin-bound αIIbβ3 predominantly exists in a fully extended, headpiece open conformation. Our results show that membrane-embedded, full-length integrin adopts an extended and open conformation when bound to its physiological macromolecular ligand. 相似文献
16.
Structural topology plays an important role in protein mechanical stability. Proteins with β-sandwich topology consisting of Greek key structural motifs, for example, I27 of muscle titin and 10FNIII of fibronectin, are mechanically resistant as shown by single-molecule force spectroscopy (SMFS). In proteins with β-sandwich topology, if the terminal strands are directly connected by backbone H-bonding then this geometry can serve as a “mechanical clamp”. Proteins with this geometry are shown to have very high unfolding forces. Here, we set out to explore the mechanical properties of a protein, M-crystallin, which belongs to β-sandwich topology consisting of Greek key motifs but its overall structure lacks the “mechanical clamp” geometry at the termini. M-crystallin is a Ca2+ binding protein from Methanosarcina acetivorans that is evolutionarily related to the vertebrate eye lens β and γ-crystallins. We constructed an octamer of crystallin, (M-crystallin)8, and using SMFS, we show that M-crystallin unfolds in a two-state manner with an unfolding force ∼90 pN (at a pulling speed of 1000 nm/sec), which is much lower than that of I27. Our study highlights that the β-sandwich topology proteins with a different strand-connectivity than that of I27 and 10FNIII, as well as lacking “mechanical clamp” geometry, can be mechanically resistant. Furthermore, Ca2+ binding not only stabilizes M-crystallin by 11.4 kcal/mol but also increases its unfolding force by ∼35 pN at the same pulling speed. The differences in the mechanical properties of apo and holo M-crystallins are further characterized using pulling speed dependent measurements and they show that Ca2+ binding reduces the unfolding potential width from 0.55 nm to 0.38 nm. These results are explained using a simple two-state unfolding energy landscape. 相似文献
17.
Platelet activation must be tightly controlled to provide an effective, but not excessive, response to vascular injury. Cytosolic calcium is a critical regulator of platelet function, including granule secretion, integrin activation, and phosphatidylserine (PS) exposure. Here we report that the novel protein kinase C isoform, PKCθ, plays an important role in negatively regulating Ca2+ signaling downstream of the major collagen receptor, glycoprotein VI (GPVI). This limits PS exposure and so may prevent excessive platelet procoagulant activity. Stimulation of GPVI resulted in significantly higher and more sustained Ca2+ signals in PKCθ−/− platelets. PKCθ acts at multiple distinct sites. PKCθ limits secretion, reducing autocrine ADP signaling that enhances Ca2+ release from intracellular Ca2+ stores. PKCθ thereby indirectly regulates activation of store-operated Ca2+ entry. However, PKCθ also directly and negatively regulates store-independent Ca2+ entry. This pathway, activated by the diacylglycerol analogue, 1-oleoyl-2-acetyl-sn-glycerol, was enhanced in PKCθ−/− platelets, independently of ADP secretion. Moreover, LOE-908, which blocks 1-oleoyl-2-acetyl-sn-glycerol-induced Ca2+ entry but not store-operated Ca2+ entry, blocked the enhanced GPVI-dependent Ca2+ signaling and PS exposure seen in PKCθ−/− platelets. We propose that PKCθ normally acts to restrict store-independent Ca2+ entry during GPVI signaling, which results in reduced PS exposure, limiting platelet procoagulant activity during thrombus formation. 相似文献
18.
Abdirahman M. Jama Jon Fenton Saralili D. Robertson Katalin T?r?k 《The Journal of biological chemistry》2009,284(41):28146-28155
Ca2+/calmodulin-dependent protein kinase II (αCaMKII) is thought to exert its role in memory formation by autonomous Ca2+-independent persistent activity conferred by Thr286 autophosphorylation, allowing the enzyme to remain active even when intracellular [Ca2+] has returned to resting levels. Ca2+ sequestration-induced inhibition, caused by a burst of Thr305/306 autophosphorylation via calmodulin (CaM) dissociation from the Thr305/306 sites, is in conflict with this view. The processes of CaM binding, autophosphorylation, and inactivation are dissected to resolve this conflict. Upon Ca2+ withdrawal, CaM sequential domain dissociation is observed, starting with the rapid release of the first (presumed N-terminal) CaM lobe, thought to be bound at the Thr305/306 sites. The time courses of Thr305/306 autophosphorylation and inactivation, however, correlate with the slow dissociation of the second (presumed C-terminal) CaM lobe. Exposure of the Thr305/306 sites is thus not sufficient for their autophosphorylation. Moreover, Thr305/306 autophosphorylation and autoinactivation are shown to occur in the continuous presence of Ca2+ and bound Ca2+/CaM by time courses similar to those seen following Ca2+ sequestration. Our investigation of the activity and mechanisms of phospho-Thr286-αCaMKII thus shows time-dependent autoinactivation, irrespective of the continued presence of Ca2+ and CaM, allowing a very short, if any, time window for Ca2+/CaM-free phospho-Thr286-αCaMKII activity. Physiologically, the time-dependent autoinactivation mechanisms of phospho-Thr286-αCaMKII (t½ of ∼50 s at 37 °C) suggest a transient kinase activity of ∼1 min duration in the induction of long term potentiation and thus memory formation.Ca2+/calmodulin-dependent protein kinase II (αCaMKII)2 is essential in hippocampal learning and N-methyl-d-aspartate receptor-dependent synaptic plasticity, causing long term potentiation (1, 2). The exact mechanisms of αCaMKII in memory functions have not yet been identified.αCaMKII is a broad specificity Ser/Thr protein kinase, which catalyzes the phosphorylation of over 100 protein and peptide substrates in vitro (3). Uniquely, the CaMKII family possesses two distinct kinase mechanisms. The first mechanism is a “canonical” intrasubunit phosphorylation, commonly found in monomeric kinases, in which the phosphorylatable residue of the substrate bound to the helical subdomain of the catalytic domain at the active site is lined up with the terminal phosphate of ATP (4). Although there is a large number of potential “canonical” substrates for αCaMKII at the synapse (5), so far AMPA receptors have been shown to be possible physiological substrates of αCaMKII (6). For the purpose of this study, syntide 2, a commonly used peptide substrate derived from phosphorylation site 2 of glycogen synthase (7), was chosen.The second mechanism, intersubunit autophosphorylation, takes advantage of the oligomeric organization of CaMKII (8). The most important autophosphorylation site in the α isoform is Thr286, which resides in the vicinity of the autoinhibitory domain (9). Peptide substrates with homologous sequences to this region have been reported to be phosphorylated by αCaMKII. This, however, occurs with a low Vmax, and these substrates show properties of a non-competitive inhibitor with respect to phosphorylation of “canonical” substrates (10) and of Thr286 autophosphorylation itself (11). Examples of such substrates include autocamtide, a peptide substrate derived from the autoinhibitory region (12) and the NR2B subunit of the N-methyl-d-aspartate receptor, which has been identified as a potential physiological target of phospho-Thr286-αCaMKII at the postsynaptic membrane (13). The possible physiological significance of NR2B phosphorylation is not yet known. There is evidence to suggest that Thr286 autophosphorylation is required to achieve full activity of the enzyme, since the unphosphorylatable T286A mutant enzyme has much diminished activity compared with wild type enzyme (14, 15).Thr286 autophosphorylation causes CaM “trapping,” a >104-fold increase in the affinity of αCaMKII for Ca2+/CaM (16–18). At the same time, Thr286 autophosphorylation is also attributed to confer Ca2+- and CaM-independent persistent “autonomous” kinase activity to αCaMKII. However, due to the extremely high affinity of phospho-Thr286-αCaMKII for Ca2+/CaM, [Ca2+] of <10 nm is required to achieve full dissociation of Ca2+/CaM, since CaM trapping occurs by virtue of Ca2+ trapping (19). Partial activity measured upon partial Ca2+ withdrawal therefore may not always reflect Ca2+/CaM-free enzyme (9). Furthermore, the physiological resting [Ca2+] range is 50–100 nm; therefore, phospho-Thr286-αCaMKII is likely always to have residual Ca2+/CaM bound. This may be partially Ca2+-saturated CaM (19).Persistent autonomous activity conferred by Thr286 autophosphorylation is thought to enable αCaMKII to function as a memory molecule (20, 21). In contrast, however, following the development of chemical long term potentiation, rapid inactivation has also been reported (22). The extent of an autonomous activity is further obscured by the finding that Ca2+ sequestration induces a burst of autophosphorylation at residues Thr305/306, followed by a loss of activity (23). Moreover, when examined across a broad range of [Ca2+], the Ca2+/CaM dependence of phospho-Thr286-αCaMKII activity is apparent (19). It is thus vital to establish the mechanisms of activation and inactivation of αCaMKII at the molecular level in order to understand how it may function physiologically in learning and memory. To this end, it is necessary to dissect the mechanisms of Ca2+/CaM dissociation, Thr305/306 autophosphorylation, and inactivation of phospho-Thr286-αCaMKII and to establish the time window for autonomous Ca2+/CaM-independent activity. 相似文献
19.
Integrin α5β1 is a major cellular receptor for the extracellular matrix protein fibronectin and plays a fundamental role during mammalian development. A crystal structure of the α5β1 integrin headpiece fragment bound by an allosteric inhibitory antibody was determined at a 2.9-Å resolution both in the absence and presence of a ligand peptide containing the Arg-Gly-Asp (RGD) sequence. The antibody-bound β1 chain accommodated the RGD ligand with very limited structural changes, which may represent the initial step of cell adhesion mediated by nonactivated integrins. Furthermore, a molecular dynamics simulation pointed to an important role for Ca2+ in the conformational coupling between the ligand-binding site and the rest of the molecule. The RGD-binding pocket is situated at the center of a trenchlike exposed surface on the top face of α5β1 devoid of glycosylation sites. The structure also enabled the precise prediction of the acceptor residue for the auxiliary synergy site of fibronectin on the α5 subunit, which was experimentally confirmed by mutagenesis and kinetic binding assays. 相似文献
20.
Voltage/Ca2+
i-gated, large conductance K+ (BK) channels result from tetrameric association of α (slo1) subunits. In most tissues, BK protein complexes include regulatory β subunits that contain two transmembrane domains (TM1, TM2), an extracellular loop, and two short intracellular termini. Four BK β types have been identified, each presenting a rather selective tissue-specific expression profile. Thus, BK β modifies current phenotype to suit physiology in a tissue-specific manner. The smooth muscle-abundant BK β1 drastically increases the channel''s apparent Ca2+
i sensitivity. The resulting phenotype is critical for BK channel activity to increase in response to Ca2+ levels reached near the channel during depolarization-induced Ca2+ influx and myocyte contraction. The eventual BK channel activation generates outward K+ currents that drive the membrane potential in the negative direction and eventually counteract depolarization-induced Ca2+ influx. The BK β1 regions responsible for the characteristic phenotype of β1-containing BK channels remain to be identified. We used patch-clamp electrophysiology on channels resulting from the combination of smooth muscle slo1 (cbv1) subunits with smooth muscle-abundant β1, neuron-abundant β4, or chimeras constructed by swapping β1 and β4 regions, and determined the contribution of specific β1 regions to the BK phenotype. At Ca2+ levels found near the channel during myocyte contraction (10 µM), channel complexes that included chimeras having both TMs from β1 and the remaining regions (“background”) from β4 showed a phenotype (Vhalf, τact, τdeact) identical to that of complexes containing wt β1. This phenotype could not be evoked by complexes that included chimeras combining either β1 TM1 or β1 TM2 with a β4 background. Likewise, β “halves” (each including β1 TM1 or β1 TM2) resulting from interrupting the continuity of the EC loop failed to render the normal phenotype, indicating that physical connection between β1 TMs via the EC loop is also necessary for proper channel function. 相似文献