Structural basis for activation of the complement system by component C4 cleavage

An essential aspect of innate immunity is recognition of molecular patterns on the surface of pathogens or altered self through the lectin and classical pathways, two of the three well-established activation pathways of the complement system. This recognition causes activation of the MASP-2 or the C1s serine proteases followed by cleavage of the protein C4. Here we present the crystal structures of the 203-kDa human C4 and the 245-kDa C4⋅MASP-2 substrate⋅enzyme complex. When C4 binds to MASP-2, substantial conformational changes in C4 are induced, and its scissile bond region becomes ordered and inserted into the protease catalytic site in a manner canonical to serine proteases. In MASP-2, an exosite located within the CCP domains recognizes the C4 C345C domain 60 Å from the scissile bond. Mutations in C4 and MASP-2 residues at the C345C–CCP interface inhibit the intermolecular interaction and C4 cleavage. The possible assembly of the huge in vivo enzyme–substrate complex consisting of glycan-bound mannan-binding lectin, MASP-2, and C4 is discussed. Our own and prior functional data suggest that C1s in the classical pathway of complement activated by, e.g., antigen–antibody complexes, also recognizes the C4 C345C domain through a CCP exosite. Our results provide a unified structural framework for understanding the early and essential step of C4 cleavage in the elimination of pathogens and altered self through two major pathways of complement activation.     

Implications for the evolution of eukaryotic amino-terminal acetyltransferase (NAT) enzymes from the structure of an archaeal ortholog

Amino-terminal acetylation is a ubiquitous modification in eukaryotes that is involved in a growing number of biological processes. There are six known eukaryotic amino-terminal acetyltransferases (NATs), which are differentiated from one another on the basis of substrate specificity. To date, two eukaryotic NATs, NatA and NatE, have been structurally characterized, of which NatA will acetylate the α-amino group of a number of nonmethionine amino-terminal residue substrates such as serine; NatE requires a substrate amino-terminal methionine residue for activity. Interestingly, these two NATs use different catalytic strategies to accomplish substrate-specific acetylation. In archaea, where this modification is less prevalent, only one NAT enzyme has been identified. Surprisingly, this enzyme is able to acetylate NatA and NatE substrates and is believed to represent an ancestral NAT variant from which the eukaryotic NAT machinery evolved. To gain insight into the evolution of NAT enzymes, we determined the X-ray crystal structure of an archaeal NAT from Sulfolobus solfataricus (ssNAT). Through the use of mutagenesis and kinetic analysis, we show that the active site of ssNAT represents a hybrid of the NatA and NatE active sites, and we highlight features of this protein that allow it to facilitate catalysis of distinct substrates through different catalytic strategies, which is a unique characteristic of this enzyme. Taken together, the structural and biochemical data presented here have implications for the evolution of eukaryotic NAT enzymes and the substrate specificities therein.The cotranslational process of amino-terminal acetylation occurs on a majority of eukaryotic proteins and mediates many biological processes, including cellular apoptosis, enzymatic regulation, protein localization, and protein degradation (1–4). In humans, three major amino-terminal acetyltransferase (NAT) complexes, called NatA, NatB, and NatC, are responsible for modifying ∼85% of all proteins that undergo amino-terminal acetylation (5). These complexes are conserved in yeast, exist as obligate heterodimers, and are differentiated from one another on the basis of substrate specificity, which is dictated by the amino-terminal sequence of the substrate protein (5, 6). Each complex consists of a single unique catalytic subunit and an additional unique auxiliary subunit that has been shown to potentiate activity and alter substrate specificity of the enzymatic component, as well as anchor the complex to the ribosome during translation (6–11). Three additional human NAT enzymes, NatD–NatF, have also been identified, but they have a much more limited set of physiological substrates, appear to be independently active, and are not well characterized across eukaryotes (12–14).The only two eukaryotic NATs that have been structurally characterized are Schizosaccharomyces pombe NatA, consisting of the Naa10p catalytic subunit and Naa15p regulatory subunit, and human NatE, which is the independently active NAA50 enzyme (15, 16). NatA, which harbors the most diversity for substrate selection, is responsible for modifying the majority of all amino-terminally acetylated proteins, which it accomplishes by recognizing proteins with amino-terminal Ala-, Cys-, Gly-, Ser-, Thr-, or Val- residues (5). NatE/NAA50 has only one known biologically relevant substrate that contains the amino-terminal sequence Met-Leu-Gly-Pro, of which only the amino-terminal Met- is absolutely required for catalysis (14). These structures and their accompanying biochemical characterization have provided significant insight into the mechanisms of substrate specificity and catalysis used by NAT enzymes. Interestingly, although NatA and NatE both catalyze α-amino group acetylation, they use unique catalytic strategies and substantially different substrate amino-terminal residue binding pockets to achieve sequence-based substrate specificity.Although only six amino-terminally acetylated proteins have been identified in Escherichia coli, large-scale proteomics studies of three different archaeal species revealed that ∼14–29% of all proteins isolated are acetylated at their amino terminus (17–20). Analysis of the thermophilic archaea Sulfolobus solfataricus genome resulted in the identification of a protein with 37% sequence identity to human NAA10 that is believed to be the only NAT in this species (21). In agreement with this hypothesis, the same study showed that this protein is able to independently acetylate NatA and NatE substrates, as well as other amino-terminal sequences corresponding to NatB and NatC substrates in vitro (21) Furthermore, no auxiliary subunit homologs could be identified in this genome, suggesting that this archaeal species dedicates just one protein to a task that requires at least nine unique proteins in humans. This ancestral NAT variant, herein referred to as ssNAT, is therefore an ideal candidate for probing the evolution of this family of enzymes and the mechanisms they use to achieve substrate-specific acetylation. Here, we report the X-ray crystal structure of the ssNAT enzyme at 1.98 Å resolution, along with a detailed comparison of ssNAT with the NatA and NatE structures. We combine information from the structure and accompanying alignments to generate a series of ssNAT mutants that we kinetically characterized to dissect the molecular features that promote the acetylation promiscuity observed for this enzyme and to propose evolutionary events that led to a diverse family of substrate-specific monomeric and heterodimeric NATs.     

Zebrafish model for human long QT syndrome

Long QT syndrome (LQTS) is a disorder of ventricular repolarization that predisposes affected individuals to lethal cardiac arrhythmias. To date, an appropriate animal model of inherited LQTS does not exist. The zebrafish is a powerful vertebrate model used to dissect molecular pathways of cardiovascular development and disease. Because fundamental electrical properties of the zebrafish heart are remarkably similar to those of the human heart, the zebrafish may be an appropriate model for studying human inherited arrhythmias. Here we describe the molecular, cellular, and electrophysiological basis of a zebrafish mutant characterized by ventricular asystole. Genetic mapping and direct sequencing identify the affected gene as kcnh2, which encodes the channel responsible for the rapidly activating delayed rectifier K(+) current (I(Kr)). We show that complete loss of functional I(Kr) in embryonic hearts leads to ventricular cell membrane depolarization, inability to generate action potentials (APs), and disrupted calcium release. A small hyperpolarizing current restores spontaneous APs, implying wild-type function of other ionic currents critical for AP generation. Heterozygous fish manifest overt cellular and electrocardiographic evidence for delayed ventricular repolarization. Our findings provide insight into the pathogenesis of homozygous kcnh2 mutations and expand the use of zebrafish mutants as a model system to study human arrhythmias.     

Rapid Inward Current in Ischemically-Injured Subepicardial Myocytes Bordering Myocardial Infarction

Ischemically-Injured Myocytes. Introduction: To determine if collagenase-dispersed epicardial myocytes overlying myocardial infarction reproduce the same altered electrophysiology observed in intact epicardiuma, multicellular tissue preparations and enzymatically-dispersed myocytes from ischemically-injured canine subepicardium were examined 1 and 4 days after myocardial infarction. Methods and Results: The electrophysiologica changes observed with alchemic injury in enzymatic ally-dispersed myocytes were not different from changes observed in multicellular tissue preparations at I and 4 days post infarction. Is chemically-injured myocytes were depolarized versus normal myocytes at [K₀] (2.5 to 40 mM) with reduced membrane potentials also observed in injured subepicardial tissue preparations [K] (4 to 24 mM). On day 1, the reduced V_max and the prolonged recovery of V_max from inactivation were consistent with the reduced membrane potentials observed at each [K]⁺. The half-maximal V_max, maximal V_max and Boltzmann constant (k) in injured myocytes were unchanged versus normal myocytes. On day 4 postinfarction, the half-maximal V_max was shifted to a more negative membrane potential, the maximal V_max. was reduced, and k was increased in injured versus normal myocytes. Prolonged recovery from inactivation was observed with depressed membrane potentials in injured myocytes on day 4. Conclusion: Enzymatically-dispersed myocytes from ischemically-injured subepicardium closely reproduce altered cellular properties observed in multicellular tissue preparations. The data suggest that 1 day postinfarction, altered conduction and refractoriness largely result from a reduced membrane potential. At 4 days, a reduced maximal V_max a shift in the inactivation curve to more negative voltages, and prolonged recovery of V_max, from inactivation also contribute to slowed conduction and prolonged refractoriness.     

兔心肌心室间复极离散的电生理学机制研究

目的观察生理和缺血状态下兔心室间复极离散,探讨临床缺血心肌发生室性心律失常的电生理机制。方法酶解法急性分离兔心室肌细胞,采用细胞膜片钳技术,分别观察对照组(正常心室内膜动作电位时程)和缺血组(缺血心室内膜动作电位时程)在不同刺激频率下[即基础循环周长(basic cycle length,BCL)=2000、1000、500及250 ms]左右心室肌心内膜细胞的动作电位时程变化。结果对照组生理心肌的室间离散分别为(47.70±7.89)ms,(45.50±7.00)ms,(40.30±7.33)ms,(37.90±6.45)ms;缺血后心室间离散则分别为(91.90±7.67)ms,(91.40±7.62)ms,(88.60±7.78)ms,(89.20±6.91)ms。结论生理状态的心肌左右心室间存在复极离散,缺血状态下心室间复极离散增大。这种心室间的复极异质性可能是缺血心肌发生室性心律失常的电生理机制之一。     

Involvement of sodium channels and two types of calcium channels in the regulation of adrenocorticotropin release

Recent electrophysiological studies from this laboratory demonstrated that anterior lobe corticotropes exhibited a tetrodotoxin-sensitive sodium current and two types of voltage-dependent calcium currents, consisting of low threshold (transient) and high threshold (long lasting) components. The present report describes cytophysiological and cytochemical studies that used specific blockers of each of these currents to assess their role in the regulation of CRF binding and ACTH secretion and storage. Two dihydropyridines, nimodipine and the pure antagonist enantiomer (-)R202-791, which block high threshold Ca2+ channels, decreased 1 h basal release by 54-74% and CRF-mediated (5 min or 3 h) release completely. Percentages of CRF-bound cells were reduced as much as 74%; however, the inhibitory effect on percentages of CRF-bound cells could be reversed by adding 10 nM Bay K 8644, (a pure dihydropyridine agonist) with the antagonists. CdCl2, which blocks both high and low threshold calcium currents, inhibited basal and CRF-stimulated ACTH release, but only the highest concentration (0.1 mM) reduced percentages of CRF-bound cells. Involvement of the low threshold Ca2+ channels could not be proved by adding dihydropyridine antagonists with 0.1 mM CdCl2. Basal and CRF-mediated ACTH release were blocked by the potent sodium channel blocker tetrodotoxin, and the highest concentration (3 microM) reduced percentages of CRF-bound cells. Basal (1 h) and CRF-stimulated (5 min) ACTH release were also inhibited in medium containing 1 mM EGTA and no Ca2+; however, percentages of CRF-bound cells were within the normal range. Densitometric analysis of stains for ACTH showed an increase in the concentration of stain per cell after a 1-h exposure to the highest concentrations of the inhibitors or to no Ca2+ and 1 mM EGTA coupled with a significant (10%) decrease in corticotrope cell area. Finally, in the last series of tests, the Bay K 8644 agonist or arginine vasopressin were used to study mechanisms of augmentation of basal or CRF-mediated ACTH release. Bay K 8644 augmented basal release in a concentration of 1 microM and CRF-mediated release in a concentration of 100 nM or 1 microM. After pretreatment with either Bay K 8644 or arginine vasopressin (10 nM) there was a significant (30%) increase in the percentage of CRF-bound cells.(ABSTRACT TRUNCATED AT 400 WORDS)     

心脏核磁共振成像与心内电生理

透视介导的心脏内电生理术一个主要缺点为术中无法正确识别软组织及其与比邻结构关系,然而,心脏核磁共振成像介导的心内电生理术却弥补了这一缺陷.心脏核磁共振成像-心内电生理术不仅可精确地展示出心脏的三维空间形态结构,而且能直接识别心律失常病灶及观察射频消融的疗效,提高复杂心律失常手术成功率及减少并发症有重要作用.因而,心脏核磁共振成像被认为在心内电生理领域具有广泛运用前景.     

Mechanistic basis for low threshold mechanosensitivity in voltage-dependent K+ channels

Living cells respond to mechanical forces applied to their outer membrane through processes referred to as "mechanosensation". Faced with hypotonic shock, to circumvent cell lysis, bacteria open large solute-passing channels to reduce the osmotic pressure gradient. In the vascular beds of vertebrate animals blood flow is regulated directly through mechanical distention-induced opening of stretch-activated channels in smooth muscle cells. Touch sensation is thought to originate in mechanically sensitive ion channels in nerve endings, and hearing in mechanically sensitive ion channels located in specialized cells of the ear. While the ubiquity of mechanosensation in living cells is evident, the ion channels underlying the transduction events in vertebrate animals have remained elusive. Here we demonstrate through electrophysiological recordings that voltage-dependent K(+) (Kv) channels exhibit exquisite sensitivity to small (physiologically relevant in magnitude) mechanical perturbations of the cell membrane. The demonstrated mechanosensitivity is quantitatively consistent with membrane tension acting on a late-opening transition through stabilization of a dilated pore. This effect causes a shift in the voltage range over which Kv channels open as well as an increase in the maximum open probability. This mechanically induced shift could allow Kv channels and perhaps other voltage-dependent ion channels to play a role in mechanosensation.     

儿茶酚胺介导的多形性室速研究进展

儿茶酚胺介导的多形性室速是一种少见却严重的遗传性心律失常,表现为无器质性心脏病的个体在运动或激动时发生双向性、多形性室速导致发作性晕厥及进展为心室颤动导致猝死。心肌细胞肌浆网异常释放钙离子使细胞内钙离子超载引起的延迟后除极可能是儿茶酚胺介导的多形性室速发生的机制。目前已知的和儿茶酚胺介导的多形性室速相关的基因为常染色体显性遗传的RyR2（位于1q42.1-q43）和常染色体隐性遗传的CASQ2（位于1p13.3-p11）。治疗：β-阻断剂适用于所有临床症状的个体和可能有RyR2突变而没有心脏事件（晕厥）或运动试验诱发的室性心律失常等病史的个体。反复心脏骤停患者需植入式心律转复除颤器。每6至12个月随访以监测疗效。患者所有的一级亲属,都应予心脏评估。     

The transmural activation sequence in porcine and canine left ventricle is markedly different during long-duration ventricular fibrillation

Background:  Humans are more similar in transmural Purkinje and cardiac ion channel distributions to dogs than pigs. The Purkinje network in pigs is transmural but confined to the endocardium in dogs. Little is known about intramural activation during long-duration ventricular fibrillation (LDVF) given these differences. We tested the hypothesis that the transmural activation sequence is similar in sinus rhythm (SR) and LDVF in dogs as well as pigs, but different between species.
Methods and Results:  In six pigs and seven dogs, 50–60 plunge needles (six electrodes, 2-mm spacing) were placed throughout the left ventricle. Unipolar recordings were made for >10 minutes of LDVF. SR and LDVF activation times were grouped into waves by linking activations along each needle. Origin (earliest activation) and propagation direction were determined for each wave. The mean wave origin was significantly more endocardial in dogs than pigs for SR and 1 through 10 minutes of LDVF. Predominant propagation direction in LDVF and SR was endocardial to epicardial in dogs, but the opposite or equal in both directions in pigs. Fastest activation rate was epicardial in pigs, but endocardial in dogs with an increasing endocardial-to-epicardial activation rate gradient as LDVF progressed in dogs but not pigs.
Conclusions:  The transmural activation sequence in SR and LDVF is markedly different between pigs and dogs. These differences may be related to differences in Purkinje fiber and ion channel distributions and suggest that dogs are a better model for investigating activation sequences during LDVF, given the similarities with humans.     

Molecular basis for Nup37 and ELY5/ELYS recruitment to the nuclear pore complex

Nucleocytoplasmic transport is mediated by nuclear pore complexes (NPCs), enormous assemblies composed of multiple copies of ∼30 different proteins called nucleoporins. To unravel the basic scaffold underlying the NPC, we have characterized the species-specific scaffold nucleoporin Nup37 and ELY5/ELYS. Both proteins integrate directly via Nup120/160 into the universally conserved heptameric Y-complex, the critical unit for the assembly and functionality of the NPC. We present the crystal structure of Schizosaccharomyces pombe Nup37 in complex with Nup120, a 174-kDa subassembly that forms one of the two short arms of the Y-complex. Nup37 binds near the bend of the L-shaped Nup120 protein, potentially stabilizing the relative orientation of its two domains. By means of reconstitution assays, we pinpoint residues crucial for this interaction. In vivo and in vitro results show that ELY5 binds near an interface of the Nup120–Nup37 complex. Complementary biochemical and cell biological data refine and consolidate the interactions of Nup120 within the current Y-model. Finally, we propose an orientation of the Y-complex relative to the pore membrane, consistent with the lattice model.     

Effects of Milrinone on Atrioventricular Conduction in the Canine Heart Under Normal Conditions,During Atrial Flutter and After Ligation and Reperfusion of the Septal Artery

Milrinone and AV Conduction. Milrinone, in addition to its vasodilatory and inotropic actions, has been shown to enhance or restore electrical activity in depolarized tissues and to improve conduction in isolated cardiac tissues depressed by exposure to an ischemic environment. These electrophysiological actions of the drug are thought to be secondary to milrinone's ability to enhance the transmembrane calcium current (I_Ca). The present study was designed to evaluate the effectiveness of milrinone (10–100 μg/kg, IV bolus) to modulate atrioventricular (AV) nodal conduction in anesthetized dogs under normal conditions, after ligation and reperfusion of the septal coronary arteries and during atrial flutter. All dosage regimens (10–100 μg/kg milrinone) produced a positive inotropic effect with no significant changes in blood pressure. Under normal conditions, 100 jig/kg of milrinone significantly reduced the PR interval, Wenckebach cycle length and AV nodal refractory period without causing a significant change in heart rate. The improvement in AV conduction and decrease in AV nodal refractoriness led to significant dose-dependent increases in ventricular rate during atrial flutter. Low doses of milrinone also accelerated the flutter rate. Ligation of the septal coronary arteries for 2 to 6 hours produced stable atrioventricular conduction disturbances in six of the 11 dogs studied: complete AV block (n = 1), second-degree AV block (n= 2) and first-degree AV block (n= 3). Milrinone (100 μg/kg) improved AV conduction in five of the six dogs. Reperfusion of the septal arteries following 6 hours of ligation produced a sustained high degree AV block in two out of five dogs. Milrinone promptly restored 1:1 conduction in both cases. Our results suggest that milrinone may be of benefit in the treatment of some cases of AV conduction disturbance and that the drug, through its effects to facilitate AV conduction and abbreviate AV refractoriness, can lead to a significant and perhaps dangerous increase in ventricular rate in patients with atrial flutter or fibrillation. (J Cardiovasc Electrophysiol, Vol 1. pp. 93–102. 1990)     

Experimentally calibrated population of models predicts and explains intersubject variability in cardiac cellular electrophysiology

Cellular and ionic causes of variability in the electrophysiological activity of hearts from individuals of the same species are unknown. However, improved understanding of this variability is key to enable prediction of the response of specific hearts to disease and therapies. Limitations of current mathematical modeling and experimental techniques hamper our ability to provide insight into variability. Here, we describe a methodology to unravel the ionic determinants of intersubject variability exhibited in experimental recordings, based on the construction and calibration of populations of models. We illustrate the methodology through its application to rabbit Purkinje preparations, because of their importance in arrhythmias and safety pharmacology assessment. We consider a set of equations describing the biophysical processes underlying rabbit Purkinje electrophysiology, and we construct a population of over 10,000 models by randomly assigning specific parameter values corresponding to ionic current conductances and kinetics. We calibrate the model population by closely comparing simulation output and experimental recordings at three pacing frequencies. We show that 213 of the 10,000 candidate models are fully consistent with the experimental dataset. Ionic properties in the 213 models cover a wide range of values, including differences up to ±100% in several conductances. Partial correlation analysis shows that particular combinations of ionic properties determine the precise shape, amplitude, and rate dependence of specific action potentials. Finally, we demonstrate that the population of models calibrated using data obtained under physiological conditions quantitatively predicts the action potential duration prolongation caused by exposure to four concentrations of the potassium channel blocker dofetilide.Biological variability is exhibited at all levels in all organs of living organisms. It manifests itself as differences in physiological function between individuals of the same species and often more drastically by significant differences in the outcome of their exposure to pathological conditions. Thus, healthy cardiac cells of the same species and location exhibit a qualitatively similar response to a stimulus, i.e., the action potential (AP). However, significant quantitative intersubject differences exist in AP morphology and duration, which may explain the different individual response of each of the cells (and patients) to disease or drug action.The variability underlying the physiological and pathological responses of different individuals has often been ignored in experimental and theoretical research, ultimately hampering the extrapolation of results to a population level. Experimentalists often average the results obtained in different preparations to reduce experimental error, therefore also averaging out the effects of intersubject variation and resulting in an important loss of information. This averaging of experimental data is inherited by theoretical research, and, consequently, models are often developed for a “typical” behavior within a particular population (1). Therefore, whereas all experimentally measured APs are different even within a homogeneous population, a single AP model is obtained from the data, again losing all information regarding intersubject variability.In this paper, we tightly couple experimental measurements and mathematical modeling to construct and calibrate a population of cardiac electrophysiology cell models representative of physiological variability, which we then use to investigate the causes of experimentally measured variability in physiological conditions and following drug response. Our research builds on previous studies by us and others (2–4) showing the importance of mathematical methods such as model populations and sensitivity analysis in investigating the ionic determinants of intersubject variability in biological properties. Previous studies have described the construction of populations of cardiac cell models, building on the work by Marder et al. in neuroscience (5). Sarkar et al. (3) constructed populations of around 300 cardiac models by varying ionic properties in an arbitrary range, but no experimental calibration of the model population was conducted nor was the predictive capacity of the model population evaluated. Davies et al. (4) adjusted model parameters in the Hund and Rudy canine model (6) to obtain 19 different models, which were fit to specific AP recordings at 1 Hz. Given the reduced number of models and data (e.g., single frequency) considered in the study, it is unlikely that the models obtained are unique or provide coverage representative of the full experimental range.Here, we describe the construction and calibration of a population of cell models that is able to represent the variability exhibited in specific experimental recordings under physiological conditions and to predict intersubject variability in response to potassium channel block. We base our investigations on rabbit Purkinje electrophysiology, because of the importance of Purkinje fibers in lethal arrhythmias (7) and in drug testing in preclinical safety pharmacology (8). We hypothesize that intersubject variability in experimentally measured APs is primarily caused by quantitative differences in the properties of ionic currents, rather than by qualitative differences in the biophysical processes underlying the currents. The equations proposed in our previous rabbit Purkinje AP Corrias–Giles–Rodriguez (CGR) model (9) are considered as the model structure to generate over 10,000 candidate models, all sharing the same equations (i.e., the same ionic biophysical processes) as in the original CGR model, but with different parameter values for the ionic properties, randomly selected within a wide range. The cell model population is then calibrated using a set of cellular biomarkers extracted from experimental AP recordings at three pacing frequencies to capture key rate-dependent AP properties. The calibrated model population is then used to identify the ionic mechanisms determining intersubject variability in each biomarker, yielding information about the relative importance of ionic currents in the generation of the AP at each pacing frequency. Finally, we demonstrate the capacity of the model population to predict variability in the response of rabbit Purkinje fibers to drug action (using an independent dataset) and to determine the underlying ionic mechanisms. We specifically show that the calibrated cell model population quantitatively predicts the prolongation of AP duration (APD) caused by exposure to four concentrations of dofetilide, a blocker of the rapid component of the delayed rectifier potassium current . We chose block as the intervention to evaluate the predictive power of our population of models because block is the main assay required in safety pharmacology assessment, because of its importance in long QT-related arrhythmias (8). The flexibility of our methodology to construct and calibrate populations of models means it can be easily applied to other areas of biology.     

From the Cover: Goniometer-based femtosecond crystallography with X-ray free electron lasers

The emerging method of femtosecond crystallography (FX) may extend the diffraction resolution accessible from small radiation-sensitive crystals and provides a means to determine catalytically accurate structures of acutely radiation-sensitive metalloenzymes. Automated goniometer-based instrumentation developed for use at the Linac Coherent Light Source enabled efficient and flexible FX experiments to be performed on a variety of sample types. In the case of rod-shaped Cpl hydrogenase crystals, only five crystals and about 30 min of beam time were used to obtain the 125 still diffraction patterns used to produce a 1.6-Å resolution electron density map. For smaller crystals, high-density grids were used to increase sample throughput; 930 myoglobin crystals mounted at random orientation inside 32 grids were exposed, demonstrating the utility of this approach. Screening results from cryocooled crystals of β₂-adrenoreceptor and an RNA polymerase II complex indicate the potential to extend the diffraction resolution obtainable from very radiation-sensitive samples beyond that possible with undulator-based synchrotron sources.Using extremely bright, short-timescale X-ray pulses produced by X-ray free-electron lasers (XFELs), femtosecond crystallography (FX) is an emerging method that expands the structural information accessible from very small or very radiation-sensitive macromolecular crystals. Central to this method is the “diffraction before destruction” (1) process in which a still diffraction image is produced by a single X-ray pulse before significant radiation-induced electronic and atomic rearrangements occur within the crystal (1–3). At the Linac Coherent Light Source (LCLS) at SLAC, a single ∼50-fs–long X-ray pulse can expose a crystal to as many X-ray photons as a typical synchrotron beam line produces in about a second. Exposing small crystals to these intense ultrashort pulses circumvents the dose limitations of conventional X-ray diffraction experiments (4) and may produce useful data to resolutions beyond what is achievable at synchrotrons (5). This innovation provides a pathway to obtain atomic information from proteins that only form micrometer- to nanometer-sized crystals, such as many membrane proteins and large multiprotein complexes. Moreover, XFELs enable “diffraction before reduction” data collection to address another major challenge in structural enzymology by providing a means to determine catalytically accurate structures of acutely radiation-sensitive metalloenzyme active sites (6), such as high-valency reaction intermediates that may be significantly photoreduced during a single X-ray exposure at a synchrotron, even at very small doses (7–11). Furthermore, the use of short (tens of femtoseconds) X-ray pulses further complements the structural characterization of biochemical reaction processes by providing access to a time domain two to three orders of magnitude faster (12, 13) than currently accessible using synchrotrons.A single X-ray pulse from the LCLS damages the illuminated sample volume and also some of the sample in the immediate vicinity, requiring the combination of measurements from discrete volumes or units of crystalline material to obtain a complete dataset (14). The first FX experiments were carried out in vacuum at the LCLS using a gas dynamic virtual nozzle (GDVN) liquid injector (15), which delivered crystals of submicron to a few microns in size, suspended in carrier solution to a series of X-ray pulses produced at up to a 120-Hz repetition rate. These pioneering experiments demonstrated the utility of serial femtosecond crystallography (SFX) and the use of crystals of less than 5 μm in size, often termed “nanocrystals” (NCs), for macromolecular structure determination to high resolution (16, 17). As NCs may be a ubiquitous but generally overlooked outcome of commercial crystallization screens that fail to produce larger crystals (18), FX may open up many systems to crystallographic analysis. However, to develop FX into a generally applicable method, a number of challenges in the areas of sample preparation, data collection, and data processing must be overcome.Obtaining a sufficient supply of crystals in an appropriate carrier solution is a first hurdle to conducting a SFX experiment. In addition to the GDVN (2, 3, 14, 16, 17, 19, 20), other injectors such as a nanoflow electrospinning injector (21) and a lipidic cubic phase (LCP) injector (22), have been developed that have a reduced flow rate and lower sample consumption. However, because injectors deliver a continuous stream of solution containing a random distribution of crystals, and the X-ray pulses are extremely short, often only a small percentage of pulses hit a crystal and produce a useful diffraction pattern. Carrying out these experiments at room temperature avoids the difficulties associated with cryoprotection, and datasets obtained at ambient temperatures can provide insight on the functional motions of protein molecules (23). However, there are different and often more complex optimization steps associated with specific injector technologies. Solutions containing a mixture of crystal sizes may require filtering to avoid clogging in the injector nozzle, and delicate crystals may be damaged from the pressures and shear forces of the delivery process itself (24). For experiments conducted in vacuo, stream formation may be disrupted by solution bubbling, drying, or freezing as it exits the injector and enters the vacuum chamber. Drop-on-demand methods that deliver single drops containing crystals to individual X-ray pulses have the potential to significantly reduce sample consumption are in development, such as acoustic and micropiezo activated technologies, but implementation has been complicated by a variety of factors, including difficulties imposed by viscous solutions and unpredictable trajectories of drops that contain crystals of varied shapes and sizes.Here, we describe an alternative strategy for FX experiments that leverages the well-established benefits of the highly automated goniometer-based setups used at state-of-the-art microfocus synchrotron beam lines, and expands these technologies to take full advantage of the unique capabilities of XFEL sources. Key to this approach is the coupling of highly automated instrumentation with specialized sample containers and customized software for efficient data collection with minimal sample consumption. High-density sample containers, such as microfluidic chips or microcrystal traps (25) for room temperature studies or grids for experiments at cryogenic temperatures, hold samples in known locations. These sample holders enable very rapid and precise positioning of crystals into the X-ray interaction region for consistent production of diffraction patterns. To optimize data completeness and resolution, data may be collected using a range of crystal sizes with a variety of X-ray beam sizes, and different regions of larger crystals may be exposed in different orientations. When small crystals align with the sample holder in a preferred orientation, exposing each crystal at varied angles to the holder surface may take advantage of this effect to enhance completeness. When the X-ray pulse mean diameter is greater than about 8 μm or is highly attenuated, the protein crystal may remain intact after exposure to the X-ray pulse and still diffract, but usually to a lower resolution as a result of radiation damage. In these cases, it is possible to rotate the crystal and collect additional diffraction patterns to use as an aid in indexing and scaling the partially recorded reflections of the initial still diffraction pattern.     

Heart failure drug digitoxin induces calcium uptake into cells by forming transmembrane calcium channels

Digitoxin and other cardiac glycosides are important, centuries-old drugs for treating congestive heart failure. However, the mechanism of action of these compounds is still being elucidated. Calcium is known to potentiate the toxicity of these drugs, and we have hypothesized that digitoxin might mediate calcium entry into cells. We report here that digitoxin molecules mediate calcium entry into intact cells. Multimers of digitoxin molecules also are able to form calcium channels in pure planar phospholipid bilayers. These digitoxin channels are blocked by Al(3+) and La(3+) but not by Mg(2+) or the classical l-type calcium channel blocker, nitrendipine. In bilayers, we find that the chemistry of the lipid affects the kinetics of the digitoxin channel activity, but not the cation selectivity. Antibodies against digitoxin promptly neutralize digitoxin channels in both cells and bilayers. We propose that these digitoxin calcium channels may be part of the mechanism by which digitoxin and other active cardiac glycosides, such as digoxin, exert system-wide actions at and above the therapeutic concentration range.     

特发性室性心律失常的发生基质研究进展

心律失常的发生基质是目前电生理领域研究的热点问题。近期研究提示:心肌组织、心肌细胞间的缝隙连接、心脏传导系统以及自主神经系统等在特发性室性心律失常的发生和维持中起着重要作用。深入研究特发性室性心律失常的发生基质将会为一些难治性室性心律失常提供新的治疗方法,有助于从根源上治疗特发性室性心律失常。     

Structural basis for substrate activation and regulation by cystathionine beta-synthase (CBS) domains in cystathionine {beta}-synthase

The catalytic potential for H₂S biogenesis and homocysteine clearance converge at the active site of cystathionine β-synthase (CBS), a pyridoxal phosphate-dependent enzyme. CBS catalyzes β-replacement reactions of either serine or cysteine by homocysteine to give cystathionine and water or H₂S, respectively. In this study, high-resolution structures of the full-length enzyme from Drosophila in which a carbanion (1.70 Å) and an aminoacrylate intermediate (1.55 Å) have been captured are reported. Electrostatic stabilization of the zwitterionic carbanion intermediate is afforded by the close positioning of an active site lysine residue that is initially used for Schiff base formation in the internal aldimine and later as a general base. Additional stabilizing interactions between active site residues and the catalytic intermediates are observed. Furthermore, the structure of the regulatory “energy-sensing” CBS domains, named after this protein, suggests a mechanism for allosteric activation by S-adenosylmethionine.     

睾酮对钙离子通道和心脏功能的影响

睾酮对人体的全身代谢、心脏的生理和病理均有着重要的影响。较高浓度的睾酮或其慢性作用可以提高T型、L型钙离子通道的密度,较低浓度或急性作用可以阻滞T型、L型钙离子通道,缩短男性Q-Tc 间期,提高对胰岛素的敏感性及改善血脂代谢。睾酮可上调钙调节蛋白、β2受体的表达,在提高细胞内钙离子浓度的情况下,可增加钙瞬变的幅度,减少钙超载。一定浓度的睾酮可以维持血管的一定张力,改善心脏传导或扩张冠脉;减少胰岛素抵抗、代谢综合征的发生,改善心肌缺血、减少心肌细胞凋亡及纤维化,保护心脏,改善心脏收缩舒张效率。     

Broad disorder and the allosteric mechanism of myosin II regulation by phosphorylation

Double electron electron resonance EPR methods was used to measure the effects of the allosteric modulators, phosphorylation, and ATP, on the distances and distance distributions between the two regulatory light chain of myosin (RLC). Three different states of smooth muscle myosin (SMM) were studied: monomers, the short-tailed subfragment heavy meromyosin, and SMM filaments. We reconstituted myosin with nine single cysteine spin-labeled RLC. For all mutants we found a broad distribution of distances that could not be explained by spin-label rotamer diversity. For SMM and heavy meromyosin, several sites showed two heterogeneous populations in the unphosphorylated samples, whereas only one was observed after phosphorylation. The data were consistent with the presence of two coexisting heterogeneous populations of structures in the unphosphorylated samples. The two populations were attributed to an on and off state by comparing data from unphosphorylated and phosphorylated samples. Models of these two states were generated using a rigid body docking approach derived from EM [Wendt T, Taylor D, Trybus KM, Taylor K (2001) Proc Natl Acad Sci USA 98:4361-4366] (PNAS, 2001, 98:4361-4366), but our data revealed a new feature of the off-state, which is heterogeneity in the orientation of the two RLC. Our average off-state structure was very similar to the Wendt model reveal a new feature of the off state, which is heterogeneity in the orientations of the two RLC. As found previously in the EM study, our on-state structure was completely different from the off-state structure. The heads are splayed out and there is even more heterogeneity in the orientations of the two RLC.     

Noninactivating voltage-gated sodium channels in severe myoclonic epilepsy of infancy

Mutations in SCN1A, the gene encoding the brain voltage-gated sodium channel alpha(1) subunit (Na(V)1.1), are associated with at least two forms of epilepsy, generalized epilepsy with febrile seizures plus and severe myoclonic epilepsy of infancy (SMEI). We examined the functional properties of five SMEI mutations by using whole-cell patch-clamp analysis of heterologously expressed recombinant human SCN1A. Two mutations (F902C and G1674R) rendered SCN1A channels nonfunctional, and a third allele (G1749E) exhibited minimal functional alterations. However, two mutations within or near the S4 segment of the fourth repeat domain (R1648C and F1661S) conferred significant impairments in fast inactivation, including persistent, noninactivating channel activity resembling the pattern of channel dysfunction observed for alleles associated with generalized epilepsy with febrile seizures plus. Our data provide evidence for a range of SCN1A functional abnormalities in SMEI, including gain-of-function defects that were not anticipated in this disorder. Our results further indicate that a complex relationship exists between phenotype and aberrant sodium channel function in these inherited epilepsies.