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梁宋平, Dongling Li‡, Yucheng Xiao§, Xia Xu§, Xia Xiong§, Shanyun Lu‡, Zhonghua Liu§, Qi Zhu§, Meichi Wang§, Xiaocheng Gu‡, and Songping Liang§¶
THE JOURNAL OF BIOLOGICAL CHEMISTRY 279(2004)37734-37740,-0001,():
-1年11月30日
Hainantoxin-IV (HNTX-IV) can specifically inhibit the neuronal tetrodotoxin-sensitive sodium channels and defines a new class of depressant spider toxin. The se-quence of native HNTX-IV is ECLGFGKGCNPSNDQCCKSSNLVCSRKHRWCKYEI-NH2. In the present study,to obtain further insight into the primary and tertiary structural requirements of neuronal sodium channel blockers, we determined the solution structure of HNTX-IV as a typical inhibitor cystine knot motif and synthesized four mutants designed based on the predicted sites followed by structural elucidation of two inactive mutants. Pharmacological studies indicated that the S12A and R26A mutants had activities near that of native HNTX-IV, while K27A and R29A demonstrated activities reduced by 2 orders of magnitude. 1H MR analysis showed the similar molecular conformations for native HNTX-IV and four synthetic mutants. Furthermore,in the determined structures of K27A and R29A,the side chains of residues 27 and 29 were located in the identical spatial position to those of native HNTX-IV.These results suggested that residues Ser12, Arg26, Lys27,and Arg29 were not responsible for stabilizing the distinct conformation of HNTX-IV, but Lys27 and Arg29 were critical for the bioactivities. The potency reductions produced by Ala substitutions were primarily due to the direct interaction of the essential residues Lys27 and Arg29 with sodium channels rather than to a conformational change. After comparison of these structures and activities with correlated toxins, we hypothesized that residues Lys27, Arg29, His28, Lys32, Phe5, and Trp30 clustered on one face of HNTX-IV were responsible for ligand binding.
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梁宋平, Yu-Cheng Xiao, Song-Ping Liang*
Toxicon 41(2003)643-650,-0001,():
-1年11月30日
A neurotoxic peptide, named Hainantoxin-V (HNTX-V), was isolated from the venom of the Chinese bird spider Selenocosmia hainana. The complete amino acid sequence of HNTX-V has been determined by Edman degradation and found to contain 35 amino acid residues with three disulfide bonds. Under whole-cell patch-clamp mode, HNTX-V was proved to inhibit the tetrodotoxin-sensitive (TTX-S) sodium currents while it had no any effects on tetrodotoxin-resistant (TTX-R) sodium currents on adult rat dorsal root ganglion neurons. The inhibition of TTX-S sodium currents by HNTX-V was tested to be concentrate-dependent with the IC50 value of 42.3nM. It did not affect the activation and inactivation kinetics of currents and did not have the effect on the active threshold of sodium channels and the voltage of peak inward currents. However,100nM HNTX-V caused a 7.7mV hyperpolarizing shift in the voltage midpoint of steady-state sodium channel inactivation.The results indicated that HNTX-V inhibited mammalian voltage-gated sodium channels through a novel mechanism distinct from other spider toxins such as d-ACTXs, m-agatoxins I-VI which bind to receptor site three to slow the inactivation kinetics of sodium currents.
Spider, Sodium current, Whole-cell recording, Dorsal root ganglion
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梁宋平, Lingyun Huang, Bingxia Li, Chen Luo, Jinyun Xie, Ping Chen, Songping Liang
Proteomics 4(2004)235-243,-0001,():
-1年11月30日
Recently, it was found that in the gynogenetic haploid and diploid embryos of goldfish, which have exactly the same genome, the haploid condition results in obstruction of gene expression and abnormal development while the diploid embryos have normal gene expression and development. A diploid-dependent regulatory apparatus was proposed to regulate gene expression. To study the difference at the protein expression level of the embryos of haploid and diploid in development, we extracted the total proteins of both the gynogenetic haploid and diploid embryos of goldfish in the same eye formation stage. Two-dimensional polyacrylamide gel electrophoresis was used to separate proteins. The stained gel images were analyzed with the PDQUEST software.A part of protein spots that were differentially expressed in haploid and diploid embryos were identified by matrix assisted laser desorption/ionisation-time of flightmass spectrometry and database analysis. Sixteen protein spots that were absolutely different (only expressed in diploid embryos but not in haploid embryos or vice versa)and 16 protein spots that were up- and downregulated were identified unambiguously,which include some proteins that are correlative with eyes development, nerve development,developing regulation, cell differentiation, and signal transduction. The different significantly gene expression during embryos developing between diploid and haploid is demonstrated.
Goldfish embryo, Mass spectrometry, Proteome analysis, Two-dimensional gel electrophoresis
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梁宋平, Yucheng Xiao, Jianzhou Tang, Yuejun Yang, Meichi Wang, Weijun Hu, Jinyun Xie, Xiongzhi Zeng, and Songping Liang‡
THE JOURNAL OF BIOLOGICAL CHEMISTRY 279(2004)26220-26226,-0001,():
-1年11月30日
We have isolated a cardiotoxin, denoted jingzhaotoxin-III (JZTX-III), from the venom of the Chinese spider Chilobrachys jingzhao. The toxin contains 36 residues stabilized by three intracellular disulfide bridges (I-IV, II-V, and III-VI), assigned by a chemical strategy of partial reduction and sequence analysis. Cloned and sequenced using 3 -rapid amplification of cDNA ends and 5 -rapid amplification of cDNA ends, the full-length cDNA encoded a 63-residue precursor of JZTX-III. Different from other spider peptides, it contains an uncommon endoproteolytic site (-X-Ser-) anterior to mature protein and the intervening regions of 5 residues, which is the smallest in spider toxin cDNAs identified to date.Under whole cell recording, JZTX-III showed no effects on voltage-gated sodium channels (VGSCs) or calcium channels in dorsal root ganglion neurons, whereas it significantly inhibited tetrodotoxin-resistant VGSCs with an IC50 value of0.38M in rat cardiac myocytes.Different from scorpion -toxins, it caused a 10-mV depolarizing shift in the channel activation threshold. The binding site for JZTX-III on VGSCs is further suggested to be site 4 with a simple competitive assay, which at 10M eliminated the slowing currents induced by Buthus martensi Karsch I (BMK-I, scorpion-like toxin) completely.JZTX-III shows higher selectivity for VGSC isoforms than other spider toxins affecting VGSCs, and the toxin hopefully represents an important ligand for discriminating cardiac VGSC subtype.
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梁宋平, Yucheng Xiao, Songping Liang*
European Journal of Pharmacology 477(2003)1-7,-0001,():
-1年11月30日
Hainantoxin-III and hainantoxin-IV, isolated from the venom of the Chinese bird spider Seleconosmia hainana, are neurotoxic peptides composed of 33–35 residues with three disulfide bonds. Using whole-cell patch-clamp technique, we investigated their action on ionic channels of adult rat dorsal root ganglion neurons. It was found that the two toxins did not affect Ca2+channels (both high voltage activated and low voltage activated types) nor tetrodotoxin-resistant voltage-gated Na+ channels (VGSCs). However, hainantoxin-III and hainantoxin-IV strongly depressed the amplitude of tetrodotoxin-sensitive Na+ currents with IC50 values of 1.1 and 44.6nM, respectively. Both hainantoxin-III (1nM) and hainantoxin-IV (50nM) caused a hyperpolarizing shift of about 10mV in the voltage midpoint of steady-state Na+ channel inactivation, but they showed difference in the reprime kinetics of VGSCs: hainantoxin-III significantly decreased the recovery rate from inactivation at a prepulse potential of 80mV while hainantoxin-IV did not do. It is interesting to note that similar to huwentoxin-IV, the two hainantoxins did not affect the activation and inactivation kinetics of Na+ currents and at a concentration of 1AM they completely inhibited the slowing inactivation currents induced by BMK-I (toxin I from the scorpion Buthus martensi Karsch), a scorpion a-like toxin.The results indicate that hainantoxin-III and hainantoxin-IV are novel spider toxins and affect the mammal neural Na+ channels through a mechanism quite different from other spider toxins targeting the neural receptor site 3, such as y-aractoxins and A-agatoxins.
Spider toxin, Dorsal ganglion neuron, Na+, current, Patch-clamp,, whole-cell
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