Multiple-conductance channels activated by excitatory amino acids in cerebellar neurons

In the mammalian central nervous system amino acids such as L-glutamate and L-aspartate are thought to act as fast synaptic transmitters. It has been suggested that at least three pharmacologically-distinguishable types of glutamate receptor occur in central neurons and that these are selectively activated by the glutamate analogues N-methyl-D-aspartate (NMDA), quisqualate and kainate. These three receptor types would be expected to open ion channels with different conductances. Hence if agonists produce similar channel conductances this would suggest they are acting on the same receptor. Another possibility is suggested by experiments on spinal neurons, where GABA (gamma-amino butyric acid) and glycine appear to open different sub-conductance levels of one class of channel while acting on different receptors. By analogy, several types of glutamate receptor could also be linked to a single type of channel with several sub-conductance states. We have examined these possibilities in cerebellar neurons by analysing the single-channel currents activated by L-glutamate, L-aspartate, NMDA, quisqualate and kainate in excised membrane patches. All of these agonists are capable of opening channels with at least five different conductance levels, the largest being about 45-50 pS. NMDA predominantly activated conductance levels above 30 pS while quisqualate and kainate mainly activated ones below 20 pS. The presence of clear transitions between levels favours the idea that the five main levels are all sub-states of the same type of channel.     

Evidence for cooperative interactions in potassium channel gating.

Cloning and expression of voltage-activated potassium ion-channel complementary DNAs has confirmed that these channels are composed of four identical subunits, each containing a voltage sensor. It has been generally accepted that the voltage sensors must reach a permissive state through one or more conformational ('gating') transitions before the channel can open. To test whether each subunit gates independently, we have constructed cDNAs encoding four subunits on a single polypeptide chain, enabling us to specify the subunit stoichiometry. The gating of heterotetramers made up from combinations of subunits with different gating phenotypes strongly suggests that individual subunits gate cooperatively, rather than independently. Nonindependent subunit gating is consistent with measurements of the kinetics of K(+)-channel gating currents and in line with the widespread subunit cooperativity observed in other multisubunit proteins.     

A large anion-selective channel has seven conductance levels

Ion channels have generally been found to have two predominant conductance levels thought to be associated with 'open' and 'closed' states, but intermediate (subconductance) states have also been reported. We have now found that a large conductance, anion-selective channel in pulmonary alveolar epithelial cells can adopt any of six open levels of conductance that are integer multiples of 60-70 pS. The channel is usually either fully open or fully closed. The frequencies of the different conductance levels are inconsistent with the notion that there are six independent channels. We suggest that the channel consists of six conducting pathways in parallel, 'co-channels', with a shared gating mechanism that can synchronously render all of them non-conducting. Other channels with lower maximum conductance may operate in a similar way but multiple conductance levels would not easily be detected because of a less favourable signal-to-noise ratio.     

The gating mechanism of the large mechanosensitive channel MscL

The mechanosensitive channel of large conductance, MscL, is a ubiquitous membrane-embedded valve involved in turgor regulation in bacteria. The crystal structure of MscL from Mycobacterium tuberculosis provides a starting point for analysing molecular mechanisms of tension-dependent channel gating. Here we develop structural models in which a cytoplasmic gate is formed by a bundle of five amino-terminal helices (S1), previously unresolved in the crystal structure. When membrane tension is applied, the transmembrane barrel expands and pulls the gate apart through the S1-M1 linker. We tested these models by substituting cysteines for residues predicted to be near each other only in either the closed or open conformation. Our results demonstrate that S1 segments form the bundle when the channel is closed, and crosslinking between S1 segments prevents opening. S1 segments interact with M2 when the channel is open, and crosslinking of S1 to M2 impedes channel closing. Gating is affected by the length of the S1-M1 linker in a manner consistent with the model, revealing critical spatial relationships between the domains that transmit force from the lipid bilayer to the channel gate.     

Relating ligand binding to activation gating in CNGA2 channels

Cyclic nucleotide-gated (CNG) ion channels mediate sensory signal transduction in photoreceptors and olfactory cells. Structurally, CNG channels are heterotetramers composed of either two or three homologue subunits. Although it is well established that activation is a cooperative process of these subunits, it remains unknown whether the cooperativity is generated by the ligand binding, the gating, or both, and how the subunits interact. In this study, the action of homotetrameric olfactory-type CNGA2 channels was studied in inside-out membrane patches by simultaneously determining channel activation and ligand binding, using the fluorescent cGMP analogue 8-DY547-cGMP as the ligand. At concentrations of 8-DY547-cGMP < 1 microM, steady-state binding was larger than steady-state activation, whereas at higher concentrations it was smaller, generating a crossover of the steady-state relationships. Global analysis of these relationships together with multiple activation time courses following cGMP jumps showed that four ligands bind to the channels and that there is significant interaction between the binding sites. Among the binding steps, the second is most critical for channel opening: its association constant is three orders of magnitude smaller than the others and it triggers a switch from a mostly closed to a maximally open state. These results contribute to unravelling the role of the subunits in the cooperative mechanism of CNGA2 channel activation and could be of general relevance for the action of other ion channels and receptors.     

神经元突触前膜C~(2+)通道状态特性及等效电路

本文讨论了由5个子单元组成的单个 C_a~(2+)通道的状态特性,证明了其中任意两个相邻状态之间的跃迁速率与玻尔兹曼能最因子 exp(—ε/KT)有关。对具有 N 个 C_a~(2+)通道的突触前膜进行了等效电路模拟。最后,给出了当突触前膜电位为矩形脉冲时,C_a~(2+)通道启通概率P 的计算机模拟结果。     

Gated regulation of CRAC channel ion selectivity by STIM1

Two defining functional features of ion channels are ion selectivity and channel gating. Ion selectivity is generally considered an immutable property of the open channel structure, whereas gating involves transitions between open and closed channel states, typically without changes in ion selectivity. In store-operated Ca(2+) release-activated Ca(2+) (CRAC) channels, the molecular mechanism of channel gating by the CRAC channel activator, stromal interaction molecule 1 (STIM1), remains unknown. CRAC channels are distinguished by a very high Ca(2+) selectivity and are instrumental in generating sustained intracellular calcium concentration elevations that are necessary for gene expression and effector function in many eukaryotic cells. Here we probe the central features of the STIM1 gating mechanism in the human CRAC channel protein, ORAI1, and identify V102, a residue located in the extracellular region of the pore, as a candidate for the channel gate. Mutations at V102 produce constitutively active CRAC channels that are open even in the absence of STIM1. Unexpectedly, although STIM1-free V102 mutant channels are not Ca(2+)-selective, their Ca(2+) selectivity is dose-dependently boosted by interactions with STIM1. Similar enhancement of Ca(2+) selectivity is also seen in wild-type ORAI1 channels by increasing the number of STIM1 activation domains that are directly tethered to ORAI1 channels, or by increasing the relative expression of full-length STIM1. Thus, exquisite Ca(2+) selectivity is not an intrinsic property of CRAC channels but rather a tuneable feature that is bestowed on otherwise non-selective ORAI1 channels by STIM1. Our results demonstrate that STIM1-mediated gating of CRAC channels occurs through an unusual mechanism in which permeation and gating are closely coupled.     

Structural mechanism of plant aquaporin gating

Plants counteract fluctuations in water supply by regulating all aquaporins in the cell plasma membrane. Channel closure results either from the dephosphorylation of two conserved serine residues under conditions of drought stress, or from the protonation of a conserved histidine residue following a drop in cytoplasmic pH due to anoxia during flooding. Here we report the X-ray structure of the spinach plasma membrane aquaporin SoPIP2;1 in its closed conformation at 2.1 A resolution and in its open conformation at 3.9 A resolution, and molecular dynamics simulations of the initial events governing gating. In the closed conformation loop D caps the channel from the cytoplasm and thereby occludes the pore. In the open conformation loop D is displaced up to 16 A and this movement opens a hydrophobic gate blocking the channel entrance from the cytoplasm. These results reveal a molecular gating mechanism which appears conserved throughout all plant plasma membrane aquaporins.     

Inverse relationship of the durations of adjacent open and shut intervals for C1 and K channels

Ion channels in cell membranes, whether voltage-dependent or activated by ligands, make repeated transitions among open and shut states during activity. Information about the number of states and the transitional pathways between them can be obtained from the durations of open and shut intervals, as transitions to states of different lifetimes result in intervals of different mean durations. If there is only one open conformation, or state, then the durations of open intervals would be independent of the durations of adjacent shut intervals. On the other hand, if a channel has two or more open states with different mean lifetimes, and if each open state is entered directly from a different shut state with a different mean lifetime, then the open intervals should be related to the adjacent shut intervals. We now report that the durations of adjacent open and shut intervals for both a C1 channel and a large conductance Ca-activated K channel in skeletal muscle are inversely related; shorter open intervals are adjacent to longer shut intervals. These findings indicate that two or more shut states make direct transitions to two or more open states, and suggest that the lifetimes of adjacent open and shut states are inversely related.     

长距离明渠输水工程突发水污染事件的应急调控

以南水北调中线总干渠典型明渠段为例,开展长距离明渠输水工程突发水污染事件应急调控研究.采取数值模拟手段分析了不同闭闸调控方式和闭闸时间条件下渠段水流运动和污染物输移扩散规律,探讨了污染云团峰值输移距离和纵向长度计算方法,推导得出了将污染云团控制在事故渠段内的应急闭闸时间计算公式.在此基础上,提出了长距离明渠输水工程突发水污染事件的应急调控方案,并结合案例验证了方案的可行性.结果表明：针对突发水污染事件,应通过对比明渠输水工程各渠段的最大水流传播时间和将污染云团控制在事故渠段内的闭闸时间来确定闭闸调控时间,从而同步实现降低工程运行安全风险和控制污染物范围的目的.     

Structure and mechanism of the M2 proton channel of influenza A virus

The integral membrane protein M2 of influenza virus forms pH-gated proton channels in the viral lipid envelope. The low pH of an endosome activates the M2 channel before haemagglutinin-mediated fusion. Conductance of protons acidifies the viral interior and thereby facilitates dissociation of the matrix protein from the viral nucleoproteins--a required process for unpacking of the viral genome. In addition to its role in release of viral nucleoproteins, M2 in the trans-Golgi network (TGN) membrane prevents premature conformational rearrangement of newly synthesized haemagglutinin during transport to the cell surface by equilibrating the pH of the TGN with that of the host cell cytoplasm. Inhibiting the proton conductance of M2 using the anti-viral drug amantadine or rimantadine inhibits viral replication. Here we present the structure of the tetrameric M2 channel in complex with rimantadine, determined by NMR. In the closed state, four tightly packed transmembrane helices define a narrow channel, in which a 'tryptophan gate' is locked by intermolecular interactions with aspartic acid. A carboxy-terminal, amphipathic helix oriented nearly perpendicular to the transmembrane helix forms an inward-facing base. Lowering the pH destabilizes the transmembrane helical packing and unlocks the gate, admitting water to conduct protons, whereas the C-terminal base remains intact, preventing dissociation of the tetramer. Rimantadine binds at four equivalent sites near the gate on the lipid-facing side of the channel and stabilizes the closed conformation of the pore. Drug-resistance mutations are predicted to counter the effect of drug binding by either increasing the hydrophilicity of the pore or weakening helix-helix packing, thus facilitating channel opening.     

Open-to-closed transition in apo maltose-binding protein observed by paramagnetic NMR

Large-scale domain rearrangements in proteins have long been recognized to have a critical function in ligand binding and recognition, catalysis and regulation. Crystal structures have provided a static picture of the apo (usually open) and holo usually closed) states. The general question arises as to whether the apo state exists as a single species in which the closed state is energetically inaccessible and interdomain rearrangement is induced by ligand or substrate binding, or whether the predominantly open form already coexists in rapid equilibrium with a minor closed species. The maltose-binding protein (MBP), a member of the bacterial periplasmic binding protein family, provides a model system for investigating this problem because it has been the subject of extensive studies by crystallography, NMR and other biophysical techniques. Here we show that although paramagnetic relaxation enhancement (PRE) data for the sugar-bound form are consistent with the crystal structure of holo MBP, the PRE data for the apo state are indicative of a rapidly exchanging mixture (ns to mus regime) of a predominantly ( approximately 95%) open form (represented by the apo crystal structure) and a minor (approximately 5%) partially closed species. Using ensemble simulated annealing refinement against the PRE data we are able to determine a ensemble average structure of the minor apo species and show that it is distinct from the sugar-bound state.     

Nakagami-m信道下双向中继选择的中断概率分析

在并行多双向中继通信系统中,提出了一种低复杂度的中断概率最优的中继选择策略,各个中继只需利用本地信道状态信息(CSI)进行分布式的选择.在Nakagami-m信道下分析了所提策略的中断概率性能,推导了系统中断概率的闭合表达式.仿真结果显示,该中继选择策略的中断概率性能在2种信道情况下均优于其他中继选择算法,且随着中继数的增加,所提策略可充分利用多中继分集提升系统中断概率性能.     

Intracellular gate opening in Shaker K+ channels defined by high-affinity metal bridges

Voltage-gated potassium channels such as Shaker help to control electrical signalling in neurons by regulating the passage of K+ across cell membranes. Ion flow is controlled by a voltage-dependent gate at the intracellular side of the pore, formed by the crossing of four alpha-helices--the inner-pore helices. The prevailing model of gating is based on a comparison of the crystal structures of two bacterial channels--KcsA in a closed state and MthK in an open state--and proposes a hinge motion at a conserved glycine that splays the inner-pore helices wide open. We show here that two types of intersubunit metal bridge, involving cysteines placed near the bundle crossing, can occur simultaneously in the open state. These bridges provide constraints on the open Shaker channel structure, and on the degree of movement upon opening. We conclude that, unlike predictions from the structure of MthK, the inner-pore helices of Shaker probably maintain the KcsA-like bundle-crossing motif in the open state, with a bend in this region at the conserved proline motif (Pro-X-Pro) not found in the bacterial channels. A narrower opening of the bundle crossing in Shaker K+ channels may help to explain why Shaker has an approximately tenfold lower conductance than its bacterial relatives.     

基于1 bit量化的超宽带同步技术研究

为有效简化FPGA运算复杂度,降低FPGA处理时钟,在传统的滑动窗相关的基础上,结合1 bit量化方法及多径能量积累的抗多径算法,提出了一种基于1 bit量化的超宽带多路并行同步方法,在此基础上设计了FPGA实现方案.推导分析了1 bit量化同步方法对系统性能的影响,给出了信噪比损失的量化结果.仿真结果表明,在低信噪比条件下,1 bit量化方法引入2 dB的信噪比损失.在高斯信道和瑞利信道下,通过针对虚警概率和漏检概率的分析及仿真,找到最优门限范围.      

The agonist effect of dihydropyridines on Ca channels

Dihydropyridines (DHP) have great potential for clinical use because of their inotropic and vasomotor effects. The mechanism of action is unknown although Ca currents have been implicated. Here we report measurements of single channel and whole cell cardiac Ca currents after application of two DHP agonists BAY K 8644 and CGP 28 392. Whole cell Ca currents from individual myocytes were increased and the 50% effective doses (ED50) were similar to those reported for contractility in rabbit aorta and guinea pig heart and catecholamine release from cat adrenal glands. The measured ED50 was also consistent with the apparent dissociation constant (Kd) of a high affinity binding site present in cardiac sarcolemmal vesicles. We propose that the molecular basis for these results is an increase in the probability that a single Ca channel, having opened and closed, will subsequently re-open during membrane depolarization. At high concentrations of BAY K 8644 and in the presence of 96 mM Ba, different effects are observed, primarily a marked prologation of open time.     

基于STM8和nRF24L01的智能车库门控制系统设计

采用STM8单片机和无线收发模块nRF24L01实现智能小区车库门的控制,并将车库门的状态信息反馈并显示至遥控发送端的LCD液晶屏上,让用户实时了解车库门是否有效关闭,门下是否有障碍物导致库门出现故障等,该系统功耗低,灵敏度高,安全稳定,且操作方便,可用于在有无市电情况下小区车库的智能控制或传统手拉车库门改造等场合.     

MOS器件的热载流子效应

热载流子效应产生的原因，首先是在沟道强电场中形成迁移率偏低的热载流子，然后由于碰撞电离而使热载流子大量增加。热电子和热空穴能够越过界面势垒向栅氧化层发射。进入栅氧化层的热载流子，或者穿透氧化层，或者造成随时间而增加的界面态，或者造成载流子陷阱。热电子或热空穴也可以受结电场的作用而进入衬底。抑制热载流子效应的方向，一是在沟道两侧制作轻掺杂区，以降低沟道电场强度；二是对栅氧化层进行氮化处理和提高氧化温度，以改善氧化层的质量，增强抗热载流子效应的能力。     

新留水闸除险加固工程初步设计研究

新留节制分水闸地处莎车县恰热克镇境内,位于叶尔羌河西岸输水总干渠57 700处,给新留渠和托克拉渠分水。本文通过对新留节制分水闸除险加固工程设计来阐述在处理四类闸时需要注意的事项,并为以后处理类似的工程提供工程实践经验。     

并行局部概率假设密度(PHD)粒子滤波

针对概率假设密度滤波权值更新效率低下的问题, 提出一种并行局部概率假设密度粒子滤波?通过聚类将粒子按目标估计个数进行分类并添加标签,通过一步预测和跟踪门限将观测区域划分为目标存在区域和不包含目标的杂波区域,修正目标所在的局部区域杂波强度公式,独立并行地计算每个目标所在区域的局部概率假设密度?仿真结果表明,并行的局部概率假设密度粒子滤波时效性更高,误差更低?