一种快速大量纯化骨骼肌ryanodine受体的方法

Ryanodine受体（RyR1）在骨骼肌细胞的兴奋-收缩偶联过程中扮演重要角色,是肌质网快速释放Ca²⁺的通道。RyR1的结构研究不同于其功能研究,需要大量的且纯度很高的蛋白。通过运用肝素（Heparin,HP）层析与羟基磷灰石（Hydroxylapatite,HA）层析,不仅在一天的时间内就可以纯化获得毫克级的RyR1,而且RyR1的纯度达到95%以上。在透射电子显微镜下观察到RyR1是一个正方形的结构,边长大约26 nm,形态类似儿童玩具风车。这个方法获得RyR1速度快、纯度高、结构完整,为进行RyR1结构研究奠定了基础。     

Binding Site Distribution of Nuclear Transport Receptors and Transport Complexes in Single Nuclear Pore Complexes

Transport through the nuclear pore complex (NPC) involves a large channel and an abundance of binding sites for nuclear transport receptors (NTRs). However, the mechanistically important distribution of NTR-binding sites along the channel is vividly debated. In this study, we visualized binding site distributions directly by two complementary optical super-resolution methods, single-molecule microscopy and 4Pi microscopy. First, we analyzed the distribution of RanGDP because this important nuclear transport substrate has two types of binding sites at the NPC, direct and indirect, NTR-mediated sites. We found that the direct binding sites had a maximum at approximately −30 nm with regard to the NPC center, whereas the indirect transport-relevant binding sites peaked at approximately −10 nm. The 20 nm-shift could be only resolved by 4Pi microscopy because of a two to threefold improved localization precision as compared with single-molecule microscopy. Then we analyzed the distribution of the NTR Kapβ1 and a Kapβ1-based transport complex and found them to have also binding maxima at approximately −10 nm. These observations support transport models in which NTR binding sites are distributed all along the transport channel and argue against models in which the cytoplasmic entrance of the channel is surrounded by a large cloud of binding sites.     

Functional Coupling of Ryanodine Receptors to KCa Channels in Smooth Muscle Cells from Rat Cerebral Arteries

The relationship between Ca²⁺ release (“Ca²⁺ sparks”) through ryanodine-sensitive Ca²⁺ release channels in the sarcoplasmic reticulum and K_Ca channels was examined in smooth muscle cells from rat cerebral arteries. Whole cell potassium currents at physiological membrane potentials (−40 mV) and intracellular Ca²⁺ were measured simultaneously, using the perforated patch clamp technique and a laser two-dimensional (x–y) scanning confocal microscope and the fluorescent Ca²⁺ indicator, fluo-3. Virtually all (96%) detectable Ca²⁺ sparks were associated with the activation of a spontaneous transient outward current (STOC) through K_Ca channels. A small number of sparks (5 of 128) were associated with currents smaller than 6 pA (mean amplitude, 4.7 pA, at −40 mV). Approximately 41% of STOCs occurred without a detectable Ca²⁺ spark. The amplitudes of the Ca²⁺ sparks correlated with the amplitudes of the STOCs (regression coefficient 0.8; P < 0.05). The half time of decay of Ca²⁺ sparks (56 ms) was longer than the associated STOCs (9 ms). The mean amplitude of the STOCs, which were associated with Ca²⁺ sparks, was 33 pA at −40 mV. The mean amplitude of the “sparkless” STOCs was smaller, 16 pA. The very significant increase in K_Ca channel open probability (>10⁴-fold) during a Ca²⁺ spark is consistent with local Ca²⁺ during a spark being in the order of 1–100 μM. Therefore, the increase in fractional fluorescence (F/F_o) measured during a Ca²⁺ spark (mean 2.04 F/F_o or ∼310 nM Ca²⁺) appears to significantly underestimate the local Ca²⁺ that activates K_Ca channels. These results indicate that the majority of ryanodine receptors that cause Ca²⁺ sparks are functionally coupled to K_Ca channels in the surface membrane, providing direct support for the idea that Ca²⁺ sparks cause STOCs.     

Modification of the Conductance and Gating Properties of Ryanodine Receptors by Suramin

Suramin, a polysulfonated napthylurea, increases the open probability and the single-channel conductance of rabbit skeletal and sheep cardiac ryanodine receptor channels. The main mechanism for the increase in P _o is an increase in the duration of open lifetimes. The effects on conduction and gating are completely reversible and involve an interaction with the cytosolic side of the channel. 10 mm dithiothreitol had no effect on the suramin-induced increase in conductance or P _o . Therefore oxidation of sulfhydryl groups on the channels does not appear to be involved. Suramin has been used as an antagonist of ATP at P₂ purinoceptors, however, we find that suramin does not antagonize the effect of ATP at skeletal or cardiac ryanodine receptor channels. The unusual gating kinetics induced by suramin suggest that it does not interact with the adenine nucleotide binding site on the ryanodine receptor but rather binds at a novel site(s). The suramin-induced changes to channel gating and conduction do not prevent the characteristic modification of single-channel properties by micromolar ryanodine. Received: 19 March 1996/Revised: 5 June 1996     

Distribution and Function of Cardiac Ryanodine Receptor Clusters in Live Ventricular Myocytes

The cardiac Ca²⁺ release channel (ryanodine receptor, RyR2) plays an essential role in excitation-contraction coupling in cardiac muscle cells. Effective and stable excitation-contraction coupling critically depends not only on the expression of RyR2, but also on its distribution. Despite its importance, little is known about the distribution and organization of RyR2 in living cells. To study the distribution of RyR2 in living cardiomyocytes, we generated a knock-in mouse model expressing a GFP-tagged RyR2 (GFP-RyR2). Confocal imaging of live ventricular myocytes isolated from the GFP-RyR2 mouse heart revealed clusters of GFP-RyR2 organized in rows with a striated pattern. Similar organization of GFP-RyR2 clusters was observed in fixed ventricular myocytes. Immunofluorescence staining with the anti-α-actinin antibody (a z-line marker) showed that nearly all GFP-RyR2 clusters were localized in the z-line zone. There were small regions with dislocated GFP-RyR2 clusters. Interestingly, these same regions also displayed dislocated z-lines. Staining with di-8-ANEPPS revealed that nearly all GFP-RyR2 clusters were co-localized with transverse but not longitudinal tubules, whereas staining with MitoTracker Red showed that GFP-RyR2 clusters were not co-localized with mitochondria in live ventricular myocytes. We also found GFP-RyR2 clusters interspersed between z-lines only at the periphery of live ventricular myocytes. Simultaneous detection of GFP-RyR2 clusters and Ca²⁺ sparks showed that Ca²⁺ sparks originated exclusively from RyR2 clusters. Ca²⁺ sparks from RyR2 clusters induced no detectable changes in mitochondrial Ca²⁺ level. These results reveal, for the first time, the distribution of RyR2 clusters and its functional correlation in living ventricular myocytes.     

Molecular Mechanisms of Regulation of the Activity of Sarcoplasmic Reticulum Ca-Release Channels (Ryanodine Receptors), Muscle Fatigue, and Severin''s Phenomenon

In this short review of the literature and our own data the characteristics of structural organization of sarcoplasmic reticulum Ca-release channels (ryanodine receptors) in different types of muscles, the participation of other sarcoplasmic reticulum proteins in excitation–contraction coupling and Ca-release channel operation, and the regulation of the channel activity by endogenous low molecular weight compounds are analyzed. Special attention is given to changes that occur in muscle cells during exhausting work and to the role of sarcoplasmic reticulum Ca-release channels in the loss of muscle contractile activity during the development of fatigue. It is concluded that the protection of muscle fibers against fatigue in the presence of the histidine-containing dipeptide carnosine, called in the literature Severin's phenomenon, is primarily connected with modulation of sarcoplasmic reticulum Ca-release channel activity by carnosine.     

Time Course of Individual Ca2+ Sparks in Frog Skeletal Muscle Recorded at High Time Resolution

Discrete Ca²⁺ release events (Ca²⁺ “sparks”) were recorded in cut segments of single frog skeletal muscle fibers using a video-rate laser-scanning confocal microscope operating in line-scan mode (63 μs per line). Fibers loaded with the Ca²⁺ indicator fluo-3 were voltage clamped at a holding potential of 0 mV, briefly reprimed at −90 mV, and then strongly depolarized with a large test pulse to activate any reprimed voltage sensors. Using this high time resolution system, it was possible to record individual Ca²⁺ sparks at ∼30-fold higher time resolution than previously attained. The resulting new experimental data provides a means of characterizing the time course of fluorescence during the brief (a few milliseconds) rising phase of a spark, which was not possible with the previously used 1.5–2 ms per line confocal systems. Analysis of the time course of individual identified events indicates that fluorescence begins to rise rather abruptly at the start of the spark, continues to rise at a slightly decreasing rate to a relatively sharp peak, and then declines along a quasi-exponential time course. The mean rise time of 198 sparks was 4.7 ± 0.1 ms, and there was no correlation between rise time and peak amplitude. Average sparks constructed by temporally and spatially superimposing and summing groups of individual sparks having similar rise times gave a lower noise representation of the sparks, consistent with the time course of individual events. In theory, the rising phase of a spark provides a lower bound estimation of the time that Ca²⁺ ions are being released by the sarcoplasmic reticulum Ca²⁺ channel(s) generating the spark. The observed time course of fluorescence suggests that the Ca²⁺ release underlying a spark could continue at a fairly constant rate throughout the rising phase of the spark, and then stop rather abruptly at the time of the peak.     

Single-Molecule Imaging of mRNA Localization and Regulation during the Integrated Stress Response

1. Download high-res image (201KB)
2. Download full-size image

     

Interaction of [3H](–)-SKF-10,047 with Brain σ Receptors: Characterization and Autoradiographic Visualization

The sigma opiates differ from other opiates in their stimulatory and psychotomimetic actions. The sigma opiate [3H](-)-SKF-10,047 has been used to characterize sigma receptors in rat nervous tissue. Binding of [3H](-)-SKF-10,047 to rat brain membranes was of high affinity, saturable, and reversible. Scatchard analysis revealed the apparent interaction of this drug with two distinct binding sites characterized by affinities of 0.03 and 75 nM (5 mM Tris-HCl buffer, pH 7.4, at 4 degrees C). Competition analyses involving rank order determinations for a series of opiates and other drugs indicate that the high-affinity binding site is the mu opiate receptor. The lower-affinity site (revealed after suppression of mu and delta receptor binding) has been identified as the sigma opiate/phencyclidine receptor. In vitro autoradiography has been used to visualize neuroanatomical patterns of receptors labeled using [3H](-)-SKF-10,047 in the presence of normorphine and [D-Ala2,D-Leu5]enkephalin to block mu and delta interactions, respectively. Labeling patterns differ markedly from those for mu, delta, or kappa receptors. The highest densities (determined by quantitative autoradiography) are found in the medial portion of the nucleus accumbens, amygdaloid nucleus, hippocampal formation, central gray, locus coeruleus, and the parabrachial nuclei. Receptors in these structures could account for the stimulatory, mood-altering, and analgesic properties of the sigma opiates. Although not the most selective sigma opiate ligand, [3H](-)-SKF-10,047 binds to sigma opiate receptors in brain, and this interaction can be readily distinguished from its interactions with other classes of brain opiate receptors.     

The EF-hand Ca2+ Binding Domain Is Not Required for Cytosolic Ca2+ Activation of the Cardiac Ryanodine Receptor

Activation of the cardiac ryanodine receptor (RyR2) by elevating cytosolic Ca²⁺ is a central step in the process of Ca²⁺-induced Ca²⁺ release, but the molecular basis of RyR2 activation by cytosolic Ca²⁺ is poorly defined. It has been proposed recently that the putative Ca²⁺ binding domain encompassing a pair of EF-hand motifs (EF1 and EF2) in the skeletal muscle ryanodine receptor (RyR1) functions as a Ca²⁺ sensor that regulates the gating of RyR1. Although the role of the EF-hand domain in RyR1 function has been studied extensively, little is known about the functional significance of the corresponding EF-hand domain in RyR2. Here we investigate the effect of mutations in the EF-hand motifs on the Ca²⁺ activation of RyR2. We found that mutations in the EF-hand motifs or deletion of the entire EF-hand domain did not affect the Ca²⁺-dependent activation of [³H]ryanodine binding or the cytosolic Ca²⁺ activation of RyR2. On the other hand, deletion of the EF-hand domain markedly suppressed the luminal Ca²⁺ activation of RyR2 and spontaneous Ca²⁺ release in HEK293 cells during store Ca²⁺ overload or store overload-induced Ca²⁺ release (SOICR). Furthermore, mutations in the EF2 motif, but not EF1 motif, of RyR2 raised the threshold for SOICR termination, whereas deletion of the EF-hand domain of RyR2 increased both the activation and termination thresholds for SOICR. These results indicate that, although the EF-hand domain is not required for RyR2 activation by cytosolic Ca²⁺, it plays an important role in luminal Ca²⁺ activation and SOICR.     

Structural Properties of the Human Protease-Activated Receptor 1 Changing by a Strong Antagonist

1. Download high-res image (226KB)
2. Download full-size image

     

Molecular Properties of 5-Hydroxytryptamine3 Receptor-Type Binding Sites Purified from NG1O8-15 Cells

5-Hydroxytryptamine3 (5-HT3) receptor-type binding sites were solubilised from NG108-15 mouse neuroblastoma x rat glioma hybrid cells using five different detergents [n-octyl-beta-D-glucoside, Triton X-100, 3-[3-(cholamidopropyl)dimethylammonio]-1-propanesulphonate (CHAPS), sodium cholate, and deoxycholate] and the solubilisation efficiencies compared. The equilibrium binding, kinetic properties, and pharmacological profile of solubilised binding sites were similar to those of 5-HT3 receptor-type binding sites (5-HT3R) in membrane preparations determined using [3H]GR65630. The solubilised binding sites were purified using an affinity column constructed by coupling the high-affinity antagonist GR119566X to an Affi-Gel 15 resin. The affinity of purified 5-HT3R for [3H]-GR65630 was reduced threefold compared to the crude soluble preparation, but the pharmacological profile was similar. The sedimentation coefficient of the purified protein (11S, detergent: CHAPS) was determined by sucrose density gradient centrifugation. The apparent molecular mass of the detergent/binding site complex (370 kDa) was determined by size exclusion chromatography in the presence of n-dodecyl-beta-D-maltoside. Gel electrophoresis of the purified protein revealed bands at apparent molecular masses of 36, 40, 50, and 76 kDa. Electron microscopy of the negatively stained purified protein showed the presence of round particles of 8-9 nm diameter with a 2-nm stained pit in the centre, closely resembling the doughnut shapes described for nicotinic acetylcholine receptors.     

Ryanodine receptor mutations in arrhythmia: The continuing mystery of channel dysfunction

Mutations in RyR2 are causative of an inherited disorder which often results in sudden cardiac death. Dysfunctional channel behaviour has been the subject of many investigations varying from single channel analysis through to complex animal models. This review discusses recent advances in the field, describes the controversy surrounding the exact consequences of RyR2 mutation and how the disparate data may be reconciled. This heterogeneity of function with respect to the effects of polymorphisms, phosphorylation, cytosolic and luminal Ca²⁺ as well as inter-domain interactions may have important implications for the recent pharmaceutical therapies which have been put forward. We surmise that a comprehensive characterisation of mutations on a case-by-case basis may be beneficial for the development of specifically targeted therapies.     

Techniques for studying membrane pores

Pore-forming proteins (PFPs) are of special interest because of the association of their activity with the disruption of the membrane impermeability barrier and cell death. They generally convert from a monomeric, soluble form into transmembrane oligomers that induce the opening of membrane pores. The study of pore formation in membranes with molecular detail remains a challenging endeavor because of its highly dynamic and complex nature, usually involving diverse oligomeric structures with different functionalities. Here we discuss current methods applied for the structural and functional characterization of PFPs at the individual vesicle and cell level. We highlight how the development of high-resolution and single-molecule imaging techniques allows the analysis of the structural organization of protein oligomers and pore entities in lipid membranes.