Neuronal inhibition by the peptide FMRFamide involves opening of S K+ channels

Neurotransmitters modulate the activity of ion channels through a variety of second messengers, including cyclic AMP, cyclic GMP and the products of phosphatidylinositol breakdown. Little is known about how different transmitters acting through different second-messenger systems interact within a cell to regulate single ion channels. We here describe the reciprocal actions of serotonin and the molluscan neuropeptide, FMRFamide, on individual K+ channels in Aplysia sensory neurons. In these cells, serotonin causes prolonged all-or-none closure of a class of background conductance K+ channels (the S channels) through cAMP-dependent protein phosphorylation. Using single-channel recording, we have found that FMRFamide produces two actions on the S channels; it increases the probability of opening of the S channels via a cAMP-independent second-messenger system and it reverses the closures of S channels produced by serotonin or cAMP.     

Open channel structure of MscL and the gating mechanism of mechanosensitive channels

Mechanosensitive channels act as membrane-embedded mechano-electrical switches, opening a large water-filled pore in response to lipid bilayer deformations. This process is critical to the response of living organisms to direct physical stimulation, such as in touch, hearing and osmoregulation. Here, we have determined the structural rearrangements that underlie these events in the large prokaryotic mechanosensitive channel (MscL) using electron paramagnetic resonance spectroscopy and site-directed spin labelling. MscL was trapped in both the open and in an intermediate closed state by modulating bilayer morphology. Transition to the intermediate state is characterized by small movements in the first transmembrane helix (TM1). Subsequent transitions to the open state are accompanied by massive rearrangements in both TM1 and TM2, as shown by large increases in probe dynamics, solvent accessibility and the elimination of all intersubunit spin-spin interactions. The open state is highly dynamic, supporting a water-filled pore of at least 25 A, lined mostly by TM1. These structures suggest a plausible molecular mechanism of gating in mechanosensitive channels.     

GTP-binding proteins mediate transmitter inhibition of voltage-dependent calcium channels

The modulation of voltage-dependent calcium channels by hormones and neurotransmitters has important implications for the control of many Ca2+-dependent cellular functions including exocytosis and contractility. We made use of electrophysiological techniques, including whole-cell patch-clamp recordings from dorsal root ganglion (DRG) neurones, to demonstrate a role for GTP-binding proteins (G-proteins) as signal transducers in the noradrenaline- and gamma-aminobutyric acid (GABA)-induced inhibition of voltage-dependent calcium channels. This action of the transmitters was blocked by: (1) preincubation of the cells with pertussis toxin (a bacterial exotoxin catalysing ADP-ribosylation of G-proteins); or (2) intracellular administration of guanosine 5'-O-(2-thiodiphosphate) (GDP-beta-S), a non-hydrolysable analogue of GDP that competitively inhibits the binding of GTP to G-proteins. Our findings provide the first direct demonstration of the G-protein-mediated inhibition of voltage-dependent calcium channels by neurotransmitters. This mode of transmitter action may explain the ability of noradrenaline and GABA to presynaptically inhibit Ca2+-dependent neurosecretion from DRG sensory neurones.     

机械敏感通道蛋白的研究进展

机械敏感通道蛋白是一类在生物体内广泛分布的离子通道蛋白,参与生物体内多种正常的生理过程,其结构异常及功能失调与多种疾病的发生、发展密切相关. 研究归纳已知的几类机械敏感通道蛋白及其在生理、病理过程中发挥的作用,并对相关疾病的靶向诊断、治疗的研究进展进行综述,最后展望其在生物医学领域的应用.     

微乳电动毛细管色谱拆分氰戊菊酯对映异构体

采用β-环糊精作为手性选择剂,使用微乳电动毛细管色谱方法在12 min内成功进行氰戊菊酯对映异构体的手性拆分.对拆分的电泳条件,如缓冲溶液的组成、浓度、pH、微乳的组成及浓度、环糊精浓度等进行了讨论及优化.结果表明,在分离电压为20 kV,选用57 cm(有效长度50 cm)×75μm内径的石英毛细管,β-环糊精的浓度为30 mmol/L,缓冲溶液pH=6.5(5 mmol/L磷酸二氢钠 10 mmol/L硼砂溶液),以40 mmol/L SDS 30 mmol/L正庚烷 300 mmol/L正丁醇为微乳,在25℃分离,可使氰戊菊酯的4个对映体基线分离.     

L-酒石酸辛酯萃取拆分普萘洛尔对映体

合成了萃取拆分普萘洛尔对映体的手性选择体L-酒石酸辛酯. 研究普萘洛尔对映体在手性选择体L-酒石酸辛酯的水和1, 2-二氯乙烷两相体系中的萃取分配行为. 考察pH值、 L-酒石酸辛酯浓度和磷酸盐浓度对分配系数(K)和分离因子(α)的影响. 研究结果表明: L-酒石酸辛酯与普萘洛尔I( )-对映体比L-酒石酸辛酯与Ⅱ (-)-对映体形成更稳定的非对映体复合物. 分配系数随着pH值的增加而增加, 分离因子随着pH值升高而下降;分配系数随着L-酒石酸辛酯浓度的增大而增大, 分离因子先随浓度增大而稳定上升, 后随浓度增加反而缓慢下降;磷酸盐浓度对分配系数和分离因子也有较大影响.     

Arterial dilations in response to calcitonin gene-related peptide involve activation of K+ channels

Calcitonin gene-related peptide (CGRP) is a 37-amino-acid peptide produced by alternative processing of messenger RNA from the calcitonin gene. CGRP is one of the most potent vasodilators known. It occurs in and is released from perivascular nerves and has been detected in the blood stream, suggesting that it is important in the control of blood flow. The mechanism by which it dilates arteries is not known. Here, we report that arterial dilations in response to CGRP are partially reversed by blockers of the ATP-sensitive potassium channel (K(ATP)), glibenclamide and barium. We also show that CGRP hyperpolarizes arterial smooth muscle and that blockers of K(ATP) channels reverse this hyperpolarization. Finally, we show that CGRP opens single K+ channels in patches on single smooth muscle cells from the same arteries. We propose that activation of K(ATP) channels underlies a substantial part of the relaxation produced by CGRP.     

A C. elegans stretch receptor neuron revealed by a mechanosensitive TRP channel homologue

The nematode Caenorhabditis elegans is commonly used as a genetic model organism for dissecting integration of the sensory and motor systems. Despite extensive genetic and behavioural analyses that have led to the identification of many genes and neural circuits involved in regulating C. elegans locomotion behaviour, it remains unclear whether and how somatosensory feedback modulates motor output during locomotion. In particular, no stretch receptors have been identified in C. elegans, raising the issue of whether stretch-receptor-mediated proprioception is used by C. elegans to regulate its locomotion behaviour. Here we have characterized TRP-4, the C. elegans homologue of the mechanosensitive TRPN channel. We show that trp-4 mutant worms bend their body abnormally, exhibiting a body posture distinct from that of wild-type worms during locomotion, suggesting that TRP-4 is involved in stretch-receptor-mediated proprioception. We show that TRP-4 acts in a single neuron, DVA, to mediate its function in proprioception, and that the activity of DVA can be stimulated by body stretch. DVA both positively and negatively modulates locomotion, providing a unique mechanism whereby a single neuron can fine-tune motor activity. Thus, DVA represents a stretch receptor neuron that regulates sensory-motor integration during C. elegans locomotion.     

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.     

HPLC手性流动相添加剂法拆分巴氯芬对映体

建立了以羧甲基-β-环糊精为手性流动相添加剂,在反相液谱条件下拆分巴氯芬对映体的方法.研究了手性添加剂的种类及浓度、流动相组成、pH值及流速等因素对拆分的影响.流动相为甲醇、乙腈、水,体积比为90∶5∶5,其中含2.00g/L CM-β-CD,pH值5.95,流速0.60mL/min,检测波长231 nm,分离度为1.31.该方法简便、快速、相对偏差小.     

液相色谱测定水样中的氧氟沙星手性对映体

利用大赛路（CHIRALCEL OD-H）手性柱对氧氟沙星对映体进行分离,考察了在流动相组分、流动相添加剂和流速对手性分离的影响。结果表明,V_正己烷∶V_乙醇∶V_乙酸＝80∶20∶0.5的流动相能够使氧氟沙星得到较好的分离,左氧氟沙星和右氧氟沙星的质量浓度在0.25～5.00μg/mL范围内线性关系良好（r²=0.9997）。实际水样中该方法的最低检出限左氧氟沙星为0.28μg/L、右氧氟沙星为0.10μg/L,最低定量限左氧氟沙星为0.36μg/L、右氧氟沙星为0.21μg/L。氧氟沙星的两组浓度加标平均回收率左氧氟沙星90.1%和88.6%、右氧氟沙星为87.6%和85.4%,左氧氟沙星的相对标准偏差（RSD）为5.2%、6.4%,右氧氟沙星的RSD为4.8%、7.6%（n=3）。     

HPLC法直接拆分dracocephinsA对映异构体

建立了一种直链淀粉键合手性固定相拆分 ｄｒａｃｏｃｅｐｈｉｎｓＡ对映异构体的方法。使用 ＣｈｉｒａｌｐａｋＩＡ柱（４６ｍｍ×２５０ｍｍ,５μｍ）,流动相为正己烷 －乙酸乙酯（１０∶９０）,流速１０ｍＬ·ｍｉｎ^－１,检测波长 ２９０ｎｍ,柱温为室温。所建立的方法能有效实现 ｄｒａｃｏｃｅｐｈｉｎｓＡ两对对映异构体的同时拆分,通过 ＣＤ检测器检测结合与单一构型异构体的比对,可以确定４种异构体出峰顺序。直链淀粉键合手性固定相可以用于 ｄｒａｃｏｃｅｐｈｉｎｓＡ对映异构体的手性分离。     

4-氯二苯甲胺的单一对映体的合成

以4-氯二苯甲酮为原料,经Leuckart反应、水解得到混旋体的4-氯二苯甲胺．以酒石酸的光学异构体为拆分剂经化学拆分得到4-氯二苯甲胺的单一对映体．本法操作简单．原料价廉易得．总收率17．6%.