Role of the beta1 subunit in large-conductance Ca(2+)-activated K(+) channel gating energetics. Mechanisms of enhanced Ca(2+) sensitivity

Over the past few years, it has become clear that an important mechanism by which large-conductance Ca²⁺-activated K⁺ channel (BK_Ca) 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 BK_Ca 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 BK_Ca 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 (K_c 7.4 μM → 9.6 μM; K_o = 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 BK_Ca channel gating that may be of general use.     

Structure of collagen receptor integrin α(1)I domain carrying the activating mutation E317A

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.     

Elucidation of a Novel Extracellular Calcium-binding Site on Metabotropic Glutamate Receptor 1α (mGluR1α) That Controls Receptor Activation

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.     

��5��1 Integrin Engagement Increases Large Conductance, Ca2+-activated K+ Channel Current and Ca2+ Sensitivity through c-src-mediated Channel Phosphorylation

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.     

Dynamic Structural Changes Are Observed upon Collagen and Metal Ion Binding to the Integrin α1 I Domain

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.     

Extracellular Calcium Modulates Actions of Orthosteric and Allosteric Ligands on Metabotropic Glutamate Receptor 1α

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.     

Akt regulates L-type Ca2+ channel activity by modulating Cavα1 protein stability

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 Ca_vα1 contains highly conserved PEST sequences (signals for rapid protein degradation), and in-frame deletion of these PEST sequences results in increased Ca_vα1 protein levels. Our findings show that Akt-dependent phosphorylation of Ca_vβ2, the LTCC chaperone for Ca_vα1, antagonizes Ca_vα1 protein degradation by preventing Ca_vα1 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.     

Complete integrin headpiece opening in eight steps

Carefully soaking crystals with Arg-Gly-Asp (RGD) peptides, we captured eight distinct RGD-bound conformations of the α_IIbβ₃ 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 α_IIb, 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.     

JNK and Ceramide Kinase Govern the Biogenesis of Lipid Droplets through Activation of Group IVA Phospholipase A2

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.     

Demonstration of catch bonds between an integrin and its ligand

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.     

Evidence that Distinct States of the Integrin α6β1 Interact with Laminin and an ADAM

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).     

Phosphorylation of the Consensus Sites of Protein Kinase A on ��1D L-type Calcium Channel

The novel α_1D 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 α_1D 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 α_1D Ca²⁺ channel by generating GST fusion proteins of the intracellular loops, N terminus, proximal and distal C termini of the α₁ subunit of α_1D 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 α_1D Ca²⁺ channel. These novel findings provide new insights into the autonomic regulation of the α_1D 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 α_1C Ca²⁺ channel is expressed in the atria and ventricles of the heart (10–13), expression of α_1D 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 α_1D along with α_1C Ca²⁺ channels contribute to L-type Ca²⁺ current (I_Ca-L) and they both play important but unique roles in the physiology/pathophysiology of the heart (6–9). Compared with α_1C, α_1D L-type Ca²⁺ channel activates at a more negative voltage range and shows slower current inactivation during depolarization (14, 15). These properties may allow α_1D Ca²⁺ channel to play critical roles in SA and AV nodes function. Indeed, α_1D Ca²⁺ channel knock-out mice exhibit significant SA dysfunction and various degrees of AV block (12, 16–19).The modulation of α_1C 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 α_1D Ca²⁺ channel activity using patch clamp studies (2). However, very little is known about potential PKA phosphorylation consensus motifs on the α_1D Ca²⁺ channel. We therefore hypothesized that the C terminus of the α₁ subunit of the α_1D Ca²⁺ channel mediates its modulation by cAMP-dependent PKA pathway.     

Conformational Changes Induced in Voltage-gated Calcium Channel Cav1.2 by BayK 8644 or FPL64176 Modify the Kinetics of Secretion Independently of Ca2+ Influx

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.     

The Structure of a Full-length Membrane-embedded Integrin Bound to a Physiological Ligand

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 α_IIbβ₃, 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 α_IIbβ₃ 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 α_IIbβ₃ 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.     

Ca2+ Binding Enhanced Mechanical Stability of an Archaeal Crystallin

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.     

Protein Kinase C�� Negatively Regulates Store-independent Ca2+ Entry and Phosphatidylserine Exposure Downstream of Glycoprotein VI in Platelets

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.     

Time-dependent Autoinactivation of Phospho-Thr286-��Ca2+/Calmodulin-dependent Protein Kinase II

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 Thr^305/306 autophosphorylation via calmodulin (CaM) dissociation from the Thr^305/306 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 Thr^305/306 sites. The time courses of Thr^305/306 autophosphorylation and inactivation, however, correlate with the slow dissociation of the second (presumed C-terminal) CaM lobe. Exposure of the Thr^305/306 sites is thus not sufficient for their autophosphorylation. Moreover, Thr^305/306 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 V_max, 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 Thr^305/306, 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, Thr^305/306 autophosphorylation, and inactivation of phospho-Thr²⁸⁶-αCaMKII and to establish the time window for autonomous Ca²⁺/CaM-independent activity.     

Crystal structure of α5β1 integrin ectodomain: atomic details of the fibronectin receptor

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.     

Both Transmembrane Domains of BK β1 Subunits Are Essential to Confer the Normal Phenotype of β1-Containing BK Channels

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 (V_half, τ_act, τ_deact) 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.