新A超家族芋螺毒素Bt14.10的克隆及合成研究

目的从我国南海西沙附近海域的桶形芋螺（Conus betulinus）中克隆出新型A-超家族芋螺毒素Bt14.10,并利用化学方法合成该毒素,鉴定其二硫键连接方式。方法提取桶形芋螺毒腺管基因组DNA,基于非翻译区及内含子序列设计合成引物进行PCR,获得Bt14.10的序列。采用Fmoc-固相法合成线性肽Bt14.10,通过空气氧化折叠获得含二硫键的折叠产物,利用两步折叠法测定其二硫键连接方式。结果发现一种新的A超家族芋螺毒素DNA序列Bt14.10,其编码的成熟肽序列为CAHSVPGMHPCKCNNTC-NH2,二硫键连接方式为＂C1-C3,C2-C4＂。结论BT14.10是一种新型A超家族芋螺毒素,具有A超家族芋螺毒素较为少见的ⅪⅤ型半胱氨酸骨架结构。     

Synaptic transmission induces transient Ca2+ concentration changes in cultured myenteric neurones.

The enteric nervous system controls most of the gastrointestinal functions. We applied confocal microscopy and the Ca2+ indicator Fluo-3 as an optical approach to study synaptic activation in cultures of myenteric neurones. The optical recording of [Ca2+]i (the intracellular Ca2+ concentration) was used to monitor activation, since [Ca2+]i is crucial in the coupling between neuronal excitation and the activation of several intracellular events. Extracellular fibre tract stimulation (2 s, 30 Hz) caused a transient [Ca2+]i rise in a subset of neurones (50%). These transients lasted for 5.2 s (n=36), with an average amplitude of 3.4 +/- 1.3 times the basal concentration. The removal of extracellular Ca2+ (n=15) or the application of 10-6 M tetrodotoxin (n=16) blocked this response. The N-type Ca2+-channel blocker omega-conotoxin (5 x 10 -7M) abolished the [Ca2+]i increase, while blockade of L-type and P/Q type Ca2+ channels had no effect. Single stimuli evoked a [Ca2+]i rise in the processes. omega-conotoxin-sensitive postsynaptic events required repetitive stimulation. Cholinergic blockade did not inhibit the [Ca2+]i rise in all neurones, suggesting that, besides acetylcholine, other neurotransmitters are involved. Optical imaging of [Ca2+]i can be used to study synaptic spread of activation in enteric neuronal circuits expressed in culture.     

Developmental regulation of T‐, N‐ and L‐type calcium currents in mouse embryonic sensory neurones

We investigated the development of a low (T-type) and two high voltage-activated (N- and L-type) calcium channel currents in large diameter dorsal root ganglion neurones acutely isolated from embryonic mice using the whole-cell patch-clamp technique. The low and high voltage-activated barium currents (LVA and HVA) were identified by their distinct threshold of activation and their sensitivity to pharmacological agents, dihydropyridines and ω-conotoxin-GVIA, at embryonic day 13 (E13), E15 and E17–18, respectively, before, during and after synaptogenesis. The amplitude and density of LVA currents, measured during a –40 mV pulse from a holding potential of –100 mV, increased significantly between E13 and E15, and remained constant between E15 and E17–18. The density of global HVA current, elicited by 0 mV pulse, increased between E13 and E15/E17–18. The density of the N-type current studied by the application of ω-conotoxin-GVIA (1 μm ) increased significantly between E13 and E15/E17–18. The use of the dihydropyridine nitrendipine (1 μm ) revealed that the density of L-type current remained constant at each stage of development. Nevertheless, application of dihydropyridine Bay K 8644 (3 μm ) demonstrated a significant slowing of the deactivation tail current between embryonic days 13 and 15, which may reflect a qualitative maturation of this class of calcium channel current. The temporal relationship between the changes in calcium channel pattern and the period of target innervation suggests possible roles of T-, N- and L-type currents during developmental key events such as natural neurone death and onset of synapse formation.     

Synthesis and liposome encapsulation of a novel 18F‐conjugate of ω‐conotoxin GVIA for the potential imaging of N‐type Ca2+ channels in the brain by positron emission tomography

ω‐Conotoxin GVIA is a potent, irreversible antagonist of N‐type voltage gated Ca²⁺ channels. A radiofluorinated analogue of GVIA could be useful in assessing regional synaptic density of the brain, in vivo, using positron emission tomography. N‐hydroxy succinimidyl 4‐[¹⁸F]fluorobenzoate was employed to site‐specifically label GVIA, preserving native binding affinity. The tracer was characterized with MALDI‐TOF mass spectrometry and colorimetric protein assay. Radiochemical decay‐corrected yield of the lysine‐24 labeled analogue of [¹⁸F]GVIA was 5%. Specific activity of this species was determined to be 1.2 × 10⁵ Ci/mmol. Encapsulation of the tracer in sulfatide containing liposomes, a potential method for enhancing blood–brain penetrance, was accomplished with 40% efficiency. Copyright © 2006 John Wiley & Sons, Ltd.     

A-超家族芋螺毒素基因的克隆及其内含子遗传进化分析

目的从中国南海独特芋螺中克隆新的A-超家族芋螺毒素基因,并分析其内含子的遗传进化关系。方法以独特芋螺基因组DNA为模板,根据A-超家族芋螺毒素基因的信号肽保守序列和3’-非翻译区(3’-UTR)保守序列设计特异性引物,通过PCR方法扩增出目的基因片段,并将其连接到pMDTM19-T载体,转化到DH5α感受态细胞并测序,同时对新的A-超家族芋螺毒素基因内含子进行遗传进化分析。结果从海南产独特芋螺基因组DNA中克隆到4条新的完整A-超家族芋螺毒素基因。CaI-M1a,CaI-M1b,CaI-M1c是编码同一种芋螺毒素CaI-M1的3个基因,它们的内含子序列存在较大的差异,特别是其中的GT重复不同。CaXXVII-M2编码产生的成熟肽含有5个半胱氨酸,与传统的属于A-超家族的α-芋螺毒素含有4个半胱氨酸的模式不同。结论首次在独特芋螺中克隆到4个新的含有完整内含子的A-超家族芋螺毒素基因,并表明其内含子的多样性与物种进化和捕食习性有一定的联系。     

Characterization of two neuronal subclasses through constellation pharmacology

Different types of neurons diverge in function because they express their own unique set or constellation of signaling molecules, including receptors and ion channels that work in concert. We describe an approach to identify functionally divergent neurons within a large, heterogeneous neuronal population while simultaneously investigating specific isoforms of signaling molecules expressed in each. In this study we characterized two subclasses of menthol-sensitive neurons from cultures of dissociated mouse dorsal-root ganglia. Although these neurons represent a small fraction of the dorsal-root ganglia neuronal population, we were able to identify them and investigate the cell-specific constellations of ion channels and receptors functionally expressed in each subclass, using a panel of selective pharmacological tools. Differences were found in the functional expression of ATP receptors, TRPA1 channels, voltage-gated calcium-, potassium-, and sodium channels, and responses to physiologically relevant cold temperatures. Furthermore, the cell-specific responses to various stimuli could be altered through pharmacological interventions targeted to the cell-specific constellation of ion channels expressed in each menthol-sensitive subclass. In fact, the normal responses to cold temperature could be reversed in the two neuronal subclasses by the coapplication of the appropriate combination of pharmacological agents. This result suggests that the functionally integrated constellation of signaling molecules in a particular type of cell is a more appropriate target for effective pharmacological intervention than a single signaling molecule. This shift from molecular to cellular targets has important implications for basic research and drug discovery. We refer to this paradigm as "constellation pharmacology."     

Insights into the origins of fish hunting in venomous cone snails from studies of Conus tessulatus

Prey shifts in carnivorous predators are events that can initiate the accelerated generation of new biodiversity. However, it is seldom possible to reconstruct how the change in prey preference occurred. Here we describe an evolutionary “smoking gun” that illuminates the transition from worm hunting to fish hunting among marine cone snails, resulting in the adaptive radiation of fish-hunting lineages comprising ∼100 piscivorous Conus species. This smoking gun is δ-conotoxin TsVIA, a peptide from the venom of Conus tessulatus that delays inactivation of vertebrate voltage-gated sodium channels. C. tessulatus is a species in a worm-hunting clade, which is phylogenetically closely related to the fish-hunting cone snail specialists. The discovery of a δ-conotoxin that potently acts on vertebrate sodium channels in the venom of a worm-hunting cone snail suggests that a closely related ancestral toxin enabled the transition from worm hunting to fish hunting, as δ-conotoxins are highly conserved among fish hunters and critical to their mechanism of prey capture; this peptide, δ-conotoxin TsVIA, has striking sequence similarity to these δ-conotoxins from piscivorous cone snail venoms. Calcium-imaging studies on dissociated dorsal root ganglion (DRG) neurons revealed the peptide’s putative molecular target (voltage-gated sodium channels) and mechanism of action (inhibition of channel inactivation). The results were confirmed by electrophysiology. This work demonstrates how elucidating the specific interactions between toxins and receptors from phylogenetically well-defined lineages can uncover molecular mechanisms that underlie significant evolutionary transitions.Among the key evolutionary events that can lead to the rapid generation of new biodiversity are shifts in food resource utilization. For biodiverse lineages, such events can trigger adaptive radiations leading to many new species. Such events may be responsible for much of the total biodiversity on Earth. For example, the bees (Hymenoptera, Apoidea, Apiformes) are a successful and ecologically important radiation with more than 16,000 named species (1). They are derived from the sphecoid wasps (Apoidea, Spheciformes), which feed arthropod prey to their developing larvae. The bees, which feed pollen to their offspring, radiated with the angiosperms. In most respects, most bee species retain sphecoid ways of life, and many are often mistaken for wasps by casual observers. The shift from larval carnivory to larval vegetarianism in bees entailed many behavioral and physiological adaptations, but these evolved so long ago that there is no practical way to reconstruct the sequence of critical events.However, more recent shifts in food resource utilization provide opportunities to study accompanying molecular changes. For example, ruminant mammals and birds have recruited originally defensive lysozymes and ribonucleases to new roles as digestive enzymes, with similar patterns of amino acid sequence changes in several independent cases (2–7). As herbivores, bees and ruminants face metabolic and other physiological challenges involved in the utilization of their food sources, which are easy to find and to harvest. By contrast, predators that shift onto a new kind of prey may often find the new resource easy to assimilate but initially very challenging to capture.The detailed mechanisms that accompany a prey shift and subsequently lead to an adaptive radiation are generally difficult to reconstruct. Molecular data from diverse species provide an opportunity to reevaluate potential mechanisms that have resulted in these pivotal events in evolution and make it possible to attempt more detailed reconstructions. In this article, we focus on an unusual and dramatic prey shift among the marine snails that hunt fish (8) and provide evidence for an evolutionary “smoking gun” supporting a specific mechanism (9). It would seem a priori a very unlikely evolutionary outcome for a relatively slow moving snail, which is unable to swim, to successfully specialize in hunting fish as prey. Nevertheless, there appear to be more than 100 species of predatory gastropod molluscs belonging to the genus Conus (cone snails) that prey on teleost fish (10–16).All cone snails, including those that hunt fish, use venom as the primary weapon for capturing their prey. Some fish-hunting cone snails have also evolved an anatomical specialization: a hollow harpoon-shaped radular tooth that allows them to tether the fish as venom is injected. In this article, we present evidence, both behavioral and molecular, that elucidates how the shift to fish hunting may have occurred in cone snails, a biodiverse lineage of molluscs comprising over 700 species.Phylogenetic data of all types are consistent with the conclusion that the ancestral cone snails preyed on marine worms. The great majority of living cone snail species are believed to feed primarily on polychaetes, although a few species will feed on other types of marine worms (e.g., hemichordates, eunicids) (17). It has been suggested (9, 18, 19) that in the Miocene, there were at least two separate events that independently triggered the generation of fish-hunting cone snail lineages from worm-hunting ancestors (another significant prey shift also occurred, namely from worm hunting to mollusc hunting). The prey shifts from worm hunting to fish hunting and mollusc hunting led to a series of robust adaptive radiations that continues to the present. The fossil evidence (15, 16) suggests that there are probably a larger number of living fish-hunting and mollusc-hunting cone snail species than there have ever been in the geologic past.     

Effects of modification at the fifth residue of μ‐conotoxin GIIIA with bulky tags on the electrically stimulated contraction of the rat diaphragm

Abstract: μ‐Conotoxin GIIIA, a peptide toxin from the cone snail, blocks muscle‐type sodium channels. Thr‐5 of μ‐conotoxin GIIIA, located on the opposite side of the active site in the globular molecule, was replaced by Cys to which the bulky tags were attached. The tagged μ‐conotoxin GIIIA derivatives, except for the phospholipid‐tagged one, exerted the biological activity with a potency slightly weaker than natural μ‐conotoxin GIIIA. When the biotinylated tags of various lengths were added, the presence of avidin suppressed the action of the biotinylated toxins of <4 nm, but not with 5 nm. The bulky biotinylated tags are useful as a caliper to measure the depth of receptor sites in the channels.     

Heat But Not Mechanical Hypersensitivity Depends on Voltage-Gated CaV2.2 Calcium Channel Activity in Peripheral Axon Terminals Innervating Skin

Voltage-gated Ca_V2.2 calcium channels are expressed in nociceptors at presynaptic terminals, soma, and axons. Ca_V2.2 channel inhibitors applied to the spinal cord relieve pain in humans and rodents, especially during pathologic pain, but a biological function of nociceptor Ca_V2.2 channels in processing of nociception, outside presynaptic terminals in the spinal cord, is underappreciated. Here, we demonstrate that functional Ca_V2.2 channels in peripheral axons innervating skin are required for capsaicin-induced heat hypersensitivity in male and female mice. We show that Ca_V2.2 channels in TRPV1-nociceptor endings are activated by capsaicin-induced depolarization and contribute to increased intracellular calcium. Capsaicin induces hypersensitivity of both thermal nociceptors and mechanoreceptors, but only heat hypersensitivity depends on peripheral Ca_V2.2 channel activity, and especially a cell-type-specific Ca_V2.2 splice isoform. Ca_V2.2 channels at peripheral nerve endings might be important therapeutic targets to mitigate certain forms of chronic pain.SIGNIFICANCE STATEMENT It is generally assumed that nociceptor termini in the spinal cord dorsal horn are the functionally significant sites of Ca_V2.2 channel in control of transmitter release and the transmission of sensory information from the periphery to central sites. We show that peripheral Ca_V2.2 channels are essential for the classic heat hypersensitivity response to develop in skin following capsaicin exposure. This function of Ca_V2.2 is highly selective for heat, but not mechanical hypersensitivity induced by capsaicin exposure, and is not a property of closely related Ca_V2.1 channels. Our findings suggest that interrupting Ca_V2.2-dependent calcium entry in skin might reduce heat hypersensitivity that develops after noxious heat exposure and may limit the degree of heat hypersensitivity associated with certain other forms of pain.