siRNA抑制Cav2.2e[37a]基因表达的研究
|
摘要
N-型钙通道是治疗慢性神经痛的一个重要药理学靶点,目前临床上应用的N型钙通道阻断剂不仅影响伤害性感受传导通路的功能,也影响中枢神经系统及自主神经系统的功能。最近有研究发现N型钙通道Car2.2e[37a]亚型仅表达于背根神经节。本研究以CaV2.2e[37a]作为靶基因,设计筛选对该基因有特异性抑制作用的siRNA,并研究该siRNA对CaV2.2e[37a]基因的抑制效果,为进一步探讨用siRNA特异性抑制CaV2.2e[37a]产生的镇痛作用及研发以cav2.2e[37a]为靶点的镇痛药物提供实验依据。
第一部分EGFP-e37a/e37b融合基因质粒的构建及e37a细胞株的建立
目的:为筛选特异性抑制Car2.2e[37a】表达的siRNA,通过构建融合基因EGFP-e37a和EGFP-e37b作为模拟靶基因,建立筛选siRNA的技术平台。方法:①表达EGFP-e37a和EGFP-e37b融合基因质粒的构建:以pcDNA316-EGFP为模板,采用重叠延伸PCR的方法扩增EGFP-e37a/e37b靶基因片段。将目的片段与pMD18-T载体连接后测序,序列正确后用酶切法克隆入pAAV2-neo载体并鉴定,得到重组质粒pAAV2-neo-EGFP-e37a和pAAV2-neo-EGFP-e37b。②稳定表达EGFP-e37a融合基因的BHK细胞株的建立:将重组质粒pAAV2-neo-EGFP-e37a在脂质体作用下转染到BHK细胞表达,荧光显微镜检测;通过G418筛选后在荧光显微镜下用稀释法挑细胞单克隆,建立稳定表达EGFP-e37a融合蛋白的BHK细胞株,命名为e37a细胞株。结果:①将得到的DNA片段用PCR方法鉴定,可见在750～1000bp之间有一条清晰单一的扩增条带;对克隆到pMD18-T载体中的片段进行PCR鉴定并进行测序,将克隆入真核载体的目的基因酶切鉴定,得到与预期一致的结果;②重组质粒pAAV_2-neo-EGFP-e37a在BHK细胞中表达,荧光显微镜检测结果可见表达效率达到40-50%;挑取了7株阳性细胞克隆,经荧光显微镜检测可见BHK细胞100%表达融合蛋白EGFP-e37a。结论:利用重叠延伸PCR方法成功构建融合基因EGFP-e37a和EGFP-e37b;建立了针对Cav2.2基因外显子e37a进行RNAi的siRNA筛选平台。
第二部分抑制CaV2.2e[37a]基因表达siRNA的设计与筛选
目的:设计并构建以CaV2.2的外显子e37a为靶基因的siRNA质粒,筛选对EGFP-e37a有特异性抑制效果的siRNA。方法:①根据RNA干扰原理及siRNA设计原则设计siRNA,克隆至pGPU6/Neo-siRNA载体,分别命名为Ⅰ,Ⅱ,Ⅲ,Ⅳ;②质粒共转染进行RNAi实验:用质粒pGPU6/Neo siRNA分别与质粒pAAV_2-neo-EGFP-e37a共转染,转染质粒之比为10:1,并设质粒pAAV_2-neo-EGFP,pAAV2-neo-EGFP-e37b对照;并将pGPU6/Neo-siRNAⅡ与pAAV_2-neo-EGFP-e37a不同剂量共转染。利用荧光显微镜和流式细胞术检测干扰效果;③对e37a细胞株进行的RNAi实验:不同剂量pGPU6/Neo-siRNAⅡ转染e37a细胞株;相同剂量pGPU6/Neo-siRNAⅡ转染e37a细胞株后不同时间点(24h、48h、72h)检测干扰效果。④Western blotting检测pGPU6/Neo-siRNAⅡ对EGFP-e37a融合基因表达的干扰结果,并设EGFP-e37b、EGFP为对照。结果:①设计了4段长度为21bp的针对e37a基因的siRNA,酶切鉴定与预期一致。②质粒共转染进行RNAi抑制实验中筛选得到对EGFP-e37a有干扰作用而对EGFP-e37b无干扰作用的siRNAⅡ,干扰效率达到84-90%,siRNAⅡ与质粒pAAV2-neo-EGFP-e37a不同剂量共转染时流式细胞术检测平均荧光强度分别降低(%):10:1时为86±6.1;5:1时为82±5.9;1:1时为80±4.2;1:5时为85±6.0;1:10时为79±4.6。③对e37a细胞株进行的RNAi抑制实验中,流式细胞术检测siRNAⅡ对EGFP-e37a的干扰效率(%):3.6ug时为93±4.2,1.8ug时为95±4.9,0.9ug时为91±3.8,0.45ug时为86±5.4,0.225ug时为70±4.3.不同时间点(24h、48h、72h)的干扰效率荧光显微镜检测在24h不明显,48h抑制细胞数达到了74±4.8(%),72h抑制细胞数82±3.3(%)。④Western blotting检测:siRNAⅡ对EGFP-e37a融合基因的表达明显抑制,蛋白条带明显减弱,而对于EGFP-e37b、EGFP基因的表达没有影响。结论:本研究在BHK细胞系中采用荧光显微镜观察和流式细胞术两种方法直观而简便地筛选出特异性干扰EGFP-e37a表达的siRNAⅡ,并对其RNAi的量效和时效进行探讨,为进一步在细胞和动物水平对Cav2.2基因的C末端外显子e37a进行RNAi的研究打下基础。
第三部分特异性siRNA对CaV2.2e[37a]基因的RNAi研究
目的:检测siRNAⅡ对于Car2.2e[37a]基因表达的干扰情况。方法:①设计测序引物对四质粒进行测序。②在293FT细胞中将pGPU6/Neo-siRNAⅡ与质粒Cav2.2e[37a]共转染,Western blotting检测siRNAⅡ对基因Car2.2e[37a]表达的抑制情况,并设转染Car2.2e[37b]组、空白293FT细胞组为对照组。结果:①四质粒测序结果与Genbank序列完全一致。②Western blotting检测质粒siRNAⅡ与基因Cav2.2e[37a]转染剂量在10:1时蛋白条带消失,而对于基因Cav2.2e[37b]、空白293FT细胞的蛋白表达没有影响。结论:siRNAⅡ对基因Cav2.2e[37a]表达有特异性抑制作用,为下一步在动物活体进行RNAi实验及为探讨CaV2.2 e[37a]亚型在慢性疼痛信号转导中的作用机制奠定了基础。
N-type calcium channel is a key pharmacological target for treatment of pain, N-type calcium channel inhibitors always cause side effects.The present challenge lies in finding some ways to specifically inhibit N-type channels in the pain pathway,not only eliminating pathological pain but also maintaining normal nociception,as well as sensory and autonomic.N-type calcium channels have two isoforms,CaV2.2e[37a]and CaV2.2e[37b].All neurons express CaV2.2e[37b] but that a subset of nociceptive neurons,particularly in DRG,express CaV2.2e [37a].This study focused on specifically knockdown the expression of CaV2.2e [37a]gene by RNA interference to provide the base for exploring the N-type calcium channel isoform inhibitor.
PartⅠConstruction of EGFP-e37a/e37b fusion gene plasmids and e37a cell line
Objective:To find out siRNAs specifically inhibiting CaV2.2e[37a]without inhibiting CaV2.2e[37b]gene expression Methods:①Construction of EGFP-37a/37b fusion gene plasmids as the mimic target genes.Full length cDNA fragment of EGFP was obtained from pcDNA316-EGFP,and amplified by high fidelity PCR Polymerase using sense primer with SOE- PCR.The fragment was inserted into eukaryotic reporter vector pAAV2 -neo by cutting with EcoRⅠand BglⅢ.The fusion gene vectors,named pAAV2 -neo-EGFP-37a/37b identified by restricted endonuclease digestion and DNA sequence analysis.②Establishment of stable cell line expressed EGFP-e37a fusion gene in BHK cells.The recombinant plasmids pAAV2 -neo-EGFP- e37a was transfect into BHK cells by Lipofectamine 2000.After transfection,cells were maintained with G418.pAAV2 -neo-EGFP-37a cell line was constructed with dilution.Result:①EGFP-37a/37b were characterized by PCR and digestion by EcoRⅠand BglⅡ.The length of fragments was about 829 bp.The positive clones were subjected to sequencing and the results were shown as expected.②The efficiency of recombinant plasmids pAAV2 -neo- EGFP- e37a transfection into BHK cells was about 40-50%.7 strains positive cell clones were selected by G418.Conclusions: Successfully construction of EGFP-37a/37b fusion gene plasmid as the mimic target genes by SOE-PCR.Establishment the screening platform to screen siRNA for inhibiting CaV2.2e[37a]gene expression.
PartⅡDesigning and screening siRNA for inhibiting expression of CaV2.2e[37a]gene
Objective:To design and construct plasmids siRNA according to CaV2.2e[37a] gene and find out siRNAs specifically inhibiting EGFP-37a without inhibiting EGFP- 37b gene expression.Methods:①Following siRNA design principles, four e37a siRNAs,namedⅠ,Ⅱ,Ⅲ,Ⅳ,were synthesized and clone into vector pGPU6/Neo.②Plasmids pAAV2- neo-EGFP-e37a were co-transfected into BHK cells with pGPU6 /Neo-siRNA at ratio 10:1.Plasmids pAAV2- neo- EGFP, pAAV2-neo-EGFP-e37b was used as control.Plasmids siRNAⅡwere co-transfected into BHK cells with pAAV2- neo-EGFP-e37a at different dose.To assess the effect of RNAi on EGFP-37a fusion protein,microscopy and fluorescence activated cell sorting(FACS) were used.③RNAi in e37a cell line:Plasmids siRNAⅡwere transfected into e37a cell line at different dose and detected the effect of RNAi at different points of time.④Fusion protein expression was detected by western blotting analysis.Results:①Design 4 pairs of 21 bp siRNAs.The result of restricted endonuclease digestion results showed the inserted fragment was as expected.②In the 4 pairs of siRNA,siRNAⅡinduced a significant decrease of EGFP-e37a expression(84-90%),without interfering EGFP- 37b gene expression.Plasmids siRNAⅡwere co-transfected into BHK cells with pAAV2- neo-EGFP-e37a at different dose,and reducing the percentage of the mean fluorescence intensity(MFI) were:10:1,86±6.1(%);5:1,82±5.9 (%);1:1,80±4.2(%);1:5 85±6.0(%);1:10,79±4.6(%).③RNAi in e37a cell line:Plasmids siRNAⅡreducing the percentage of MFI were:3.6ug, 93±4.2(%);1.8ug,95±4.9(%);0.9ug,91±3.8(%);0.45ug,86±5.4(%);0.225ug, 70±4.3(%).24h after the transfection,the efficiency of RNAi was not obvious.At the time point of 48h,the efficiency of RNAi amount to 74±4.8(%);72h,82±3.3 (%).④siRNAⅡcould decrease the expression of EGFP-e37a protein level obviously,while not decrease the expression of EGFP-e37b and EGFR Conclusions:siRNAⅡthat specifically inhibiting EGFP-37a without inhibiting EGFP- 37b gene expression has been found out by fluorescence microscopy and FACS.So we provide a basis tbr studying of RNAi of Cav2.2e[37a]in vitro and in vivo.
PartⅢStudy of the specific siRNA inhibiting CaV2.2e[37a]gene expression
Objective:To detect the effect of siRNAⅡinhibiting CaV2.2e[37a]gene expression by western blotting.Methods:①Design the primer for sequencing four plasmids.②Plasmid siRNAⅡco-transfected into 293FT cells with plasmids Cav2.2e[37a],Cav2.2e[37b],β3,α2σ1 respectively,and detected the expression of Cav2.2e[37a]gene by western blotting.Result:①The results of DNA sequencing showed the four plasmids were as Genebank sequences.②The protein on band 6 diminished obviously which indicate that siRNAⅡdecrease the generation of CaV2.2e[37a]expression protein in 293FT cells.Conclusions: SiRNAⅡcould inhibit Cav2.2e[37a]gene expression specifically without inhibiting CaV2.2e[37b]gene expression,and providing a potential agent for studying RNAi Cav2.2e[37a]isoform in noxious stimuli transmission..
第一部分EGFP-e37a/e37b融合基因质粒的构建及e37a细胞株的建立
目的:为筛选特异性抑制Car2.2e[37a】表达的siRNA,通过构建融合基因EGFP-e37a和EGFP-e37b作为模拟靶基因,建立筛选siRNA的技术平台。方法:①表达EGFP-e37a和EGFP-e37b融合基因质粒的构建:以pcDNA316-EGFP为模板,采用重叠延伸PCR的方法扩增EGFP-e37a/e37b靶基因片段。将目的片段与pMD18-T载体连接后测序,序列正确后用酶切法克隆入pAAV2-neo载体并鉴定,得到重组质粒pAAV2-neo-EGFP-e37a和pAAV2-neo-EGFP-e37b。②稳定表达EGFP-e37a融合基因的BHK细胞株的建立:将重组质粒pAAV2-neo-EGFP-e37a在脂质体作用下转染到BHK细胞表达,荧光显微镜检测;通过G418筛选后在荧光显微镜下用稀释法挑细胞单克隆,建立稳定表达EGFP-e37a融合蛋白的BHK细胞株,命名为e37a细胞株。结果:①将得到的DNA片段用PCR方法鉴定,可见在750～1000bp之间有一条清晰单一的扩增条带;对克隆到pMD18-T载体中的片段进行PCR鉴定并进行测序,将克隆入真核载体的目的基因酶切鉴定,得到与预期一致的结果;②重组质粒pAAV_2-neo-EGFP-e37a在BHK细胞中表达,荧光显微镜检测结果可见表达效率达到40-50%;挑取了7株阳性细胞克隆,经荧光显微镜检测可见BHK细胞100%表达融合蛋白EGFP-e37a。结论:利用重叠延伸PCR方法成功构建融合基因EGFP-e37a和EGFP-e37b;建立了针对Cav2.2基因外显子e37a进行RNAi的siRNA筛选平台。
第二部分抑制CaV2.2e[37a]基因表达siRNA的设计与筛选
目的:设计并构建以CaV2.2的外显子e37a为靶基因的siRNA质粒,筛选对EGFP-e37a有特异性抑制效果的siRNA。方法:①根据RNA干扰原理及siRNA设计原则设计siRNA,克隆至pGPU6/Neo-siRNA载体,分别命名为Ⅰ,Ⅱ,Ⅲ,Ⅳ;②质粒共转染进行RNAi实验:用质粒pGPU6/Neo siRNA分别与质粒pAAV_2-neo-EGFP-e37a共转染,转染质粒之比为10:1,并设质粒pAAV_2-neo-EGFP,pAAV2-neo-EGFP-e37b对照;并将pGPU6/Neo-siRNAⅡ与pAAV_2-neo-EGFP-e37a不同剂量共转染。利用荧光显微镜和流式细胞术检测干扰效果;③对e37a细胞株进行的RNAi实验:不同剂量pGPU6/Neo-siRNAⅡ转染e37a细胞株;相同剂量pGPU6/Neo-siRNAⅡ转染e37a细胞株后不同时间点(24h、48h、72h)检测干扰效果。④Western blotting检测pGPU6/Neo-siRNAⅡ对EGFP-e37a融合基因表达的干扰结果,并设EGFP-e37b、EGFP为对照。结果:①设计了4段长度为21bp的针对e37a基因的siRNA,酶切鉴定与预期一致。②质粒共转染进行RNAi抑制实验中筛选得到对EGFP-e37a有干扰作用而对EGFP-e37b无干扰作用的siRNAⅡ,干扰效率达到84-90%,siRNAⅡ与质粒pAAV2-neo-EGFP-e37a不同剂量共转染时流式细胞术检测平均荧光强度分别降低(%):10:1时为86±6.1;5:1时为82±5.9;1:1时为80±4.2;1:5时为85±6.0;1:10时为79±4.6。③对e37a细胞株进行的RNAi抑制实验中,流式细胞术检测siRNAⅡ对EGFP-e37a的干扰效率(%):3.6ug时为93±4.2,1.8ug时为95±4.9,0.9ug时为91±3.8,0.45ug时为86±5.4,0.225ug时为70±4.3.不同时间点(24h、48h、72h)的干扰效率荧光显微镜检测在24h不明显,48h抑制细胞数达到了74±4.8(%),72h抑制细胞数82±3.3(%)。④Western blotting检测:siRNAⅡ对EGFP-e37a融合基因的表达明显抑制,蛋白条带明显减弱,而对于EGFP-e37b、EGFP基因的表达没有影响。结论:本研究在BHK细胞系中采用荧光显微镜观察和流式细胞术两种方法直观而简便地筛选出特异性干扰EGFP-e37a表达的siRNAⅡ,并对其RNAi的量效和时效进行探讨,为进一步在细胞和动物水平对Cav2.2基因的C末端外显子e37a进行RNAi的研究打下基础。
第三部分特异性siRNA对CaV2.2e[37a]基因的RNAi研究
目的:检测siRNAⅡ对于Car2.2e[37a]基因表达的干扰情况。方法:①设计测序引物对四质粒进行测序。②在293FT细胞中将pGPU6/Neo-siRNAⅡ与质粒Cav2.2e[37a]共转染,Western blotting检测siRNAⅡ对基因Car2.2e[37a]表达的抑制情况,并设转染Car2.2e[37b]组、空白293FT细胞组为对照组。结果:①四质粒测序结果与Genbank序列完全一致。②Western blotting检测质粒siRNAⅡ与基因Cav2.2e[37a]转染剂量在10:1时蛋白条带消失,而对于基因Cav2.2e[37b]、空白293FT细胞的蛋白表达没有影响。结论:siRNAⅡ对基因Cav2.2e[37a]表达有特异性抑制作用,为下一步在动物活体进行RNAi实验及为探讨CaV2.2 e[37a]亚型在慢性疼痛信号转导中的作用机制奠定了基础。
N-type calcium channel is a key pharmacological target for treatment of pain, N-type calcium channel inhibitors always cause side effects.The present challenge lies in finding some ways to specifically inhibit N-type channels in the pain pathway,not only eliminating pathological pain but also maintaining normal nociception,as well as sensory and autonomic.N-type calcium channels have two isoforms,CaV2.2e[37a]and CaV2.2e[37b].All neurons express CaV2.2e[37b] but that a subset of nociceptive neurons,particularly in DRG,express CaV2.2e [37a].This study focused on specifically knockdown the expression of CaV2.2e [37a]gene by RNA interference to provide the base for exploring the N-type calcium channel isoform inhibitor.
PartⅠConstruction of EGFP-e37a/e37b fusion gene plasmids and e37a cell line
Objective:To find out siRNAs specifically inhibiting CaV2.2e[37a]without inhibiting CaV2.2e[37b]gene expression Methods:①Construction of EGFP-37a/37b fusion gene plasmids as the mimic target genes.Full length cDNA fragment of EGFP was obtained from pcDNA316-EGFP,and amplified by high fidelity PCR Polymerase using sense primer with SOE- PCR.The fragment was inserted into eukaryotic reporter vector pAAV2 -neo by cutting with EcoRⅠand BglⅢ.The fusion gene vectors,named pAAV2 -neo-EGFP-37a/37b identified by restricted endonuclease digestion and DNA sequence analysis.②Establishment of stable cell line expressed EGFP-e37a fusion gene in BHK cells.The recombinant plasmids pAAV2 -neo-EGFP- e37a was transfect into BHK cells by Lipofectamine 2000.After transfection,cells were maintained with G418.pAAV2 -neo-EGFP-37a cell line was constructed with dilution.Result:①EGFP-37a/37b were characterized by PCR and digestion by EcoRⅠand BglⅡ.The length of fragments was about 829 bp.The positive clones were subjected to sequencing and the results were shown as expected.②The efficiency of recombinant plasmids pAAV2 -neo- EGFP- e37a transfection into BHK cells was about 40-50%.7 strains positive cell clones were selected by G418.Conclusions: Successfully construction of EGFP-37a/37b fusion gene plasmid as the mimic target genes by SOE-PCR.Establishment the screening platform to screen siRNA for inhibiting CaV2.2e[37a]gene expression.
PartⅡDesigning and screening siRNA for inhibiting expression of CaV2.2e[37a]gene
Objective:To design and construct plasmids siRNA according to CaV2.2e[37a] gene and find out siRNAs specifically inhibiting EGFP-37a without inhibiting EGFP- 37b gene expression.Methods:①Following siRNA design principles, four e37a siRNAs,namedⅠ,Ⅱ,Ⅲ,Ⅳ,were synthesized and clone into vector pGPU6/Neo.②Plasmids pAAV2- neo-EGFP-e37a were co-transfected into BHK cells with pGPU6 /Neo-siRNA at ratio 10:1.Plasmids pAAV2- neo- EGFP, pAAV2-neo-EGFP-e37b was used as control.Plasmids siRNAⅡwere co-transfected into BHK cells with pAAV2- neo-EGFP-e37a at different dose.To assess the effect of RNAi on EGFP-37a fusion protein,microscopy and fluorescence activated cell sorting(FACS) were used.③RNAi in e37a cell line:Plasmids siRNAⅡwere transfected into e37a cell line at different dose and detected the effect of RNAi at different points of time.④Fusion protein expression was detected by western blotting analysis.Results:①Design 4 pairs of 21 bp siRNAs.The result of restricted endonuclease digestion results showed the inserted fragment was as expected.②In the 4 pairs of siRNA,siRNAⅡinduced a significant decrease of EGFP-e37a expression(84-90%),without interfering EGFP- 37b gene expression.Plasmids siRNAⅡwere co-transfected into BHK cells with pAAV2- neo-EGFP-e37a at different dose,and reducing the percentage of the mean fluorescence intensity(MFI) were:10:1,86±6.1(%);5:1,82±5.9 (%);1:1,80±4.2(%);1:5 85±6.0(%);1:10,79±4.6(%).③RNAi in e37a cell line:Plasmids siRNAⅡreducing the percentage of MFI were:3.6ug, 93±4.2(%);1.8ug,95±4.9(%);0.9ug,91±3.8(%);0.45ug,86±5.4(%);0.225ug, 70±4.3(%).24h after the transfection,the efficiency of RNAi was not obvious.At the time point of 48h,the efficiency of RNAi amount to 74±4.8(%);72h,82±3.3 (%).④siRNAⅡcould decrease the expression of EGFP-e37a protein level obviously,while not decrease the expression of EGFP-e37b and EGFR Conclusions:siRNAⅡthat specifically inhibiting EGFP-37a without inhibiting EGFP- 37b gene expression has been found out by fluorescence microscopy and FACS.So we provide a basis tbr studying of RNAi of Cav2.2e[37a]in vitro and in vivo.
PartⅢStudy of the specific siRNA inhibiting CaV2.2e[37a]gene expression
Objective:To detect the effect of siRNAⅡinhibiting CaV2.2e[37a]gene expression by western blotting.Methods:①Design the primer for sequencing four plasmids.②Plasmid siRNAⅡco-transfected into 293FT cells with plasmids Cav2.2e[37a],Cav2.2e[37b],β3,α2σ1 respectively,and detected the expression of Cav2.2e[37a]gene by western blotting.Result:①The results of DNA sequencing showed the four plasmids were as Genebank sequences.②The protein on band 6 diminished obviously which indicate that siRNAⅡdecrease the generation of CaV2.2e[37a]expression protein in 293FT cells.Conclusions: SiRNAⅡcould inhibit Cav2.2e[37a]gene expression specifically without inhibiting CaV2.2e[37b]gene expression,and providing a potential agent for studying RNAi Cav2.2e[37a]isoform in noxious stimuli transmission..
引文
[1]CA.Reid,J.M.Bekkers,J.D.Clements.Presynaptic Ca~(2+)channels:a functional patchwork.Trends Neurosci.2003.26:683-687.
[2]K.Dunlap,J.I.Luebke.T.J.Turner.Exocytotic Ca channels in mammalian central neurons.Trends Neurosci.1995,18:89-98.
[3]Jarvis ES,Zamponi GW.Interactions between presynaptic Ca2+channels,cytoplasmic messengers and proteins of the synaptic vesicle release complex.Trends Pharmacol Sci,2001,22:519-525.
[4]Augustine G.How does calcium trigger neurotransmitter release?Curr Opin Neurobiol,2001,11:320-326.
[5]T.A.Brosenitsch,D.M.Katz,Physiological patterns of electrical stimulation can induce neuronal gene expression by activating N-type calcium channels,J.Neurosci.2001,21:2571-2579.
[6]O.T.Jones,G.M.Bernstein,E.J.Jones,et al.N-Type calcium channels in the developing rat hippocampus:subunit,complex,and regional expression,J.Neurosci.1997,17:6152-6164.
[7]Altier C,Zamponi GW.Targeting Ca~(2+)channels to treat pain:T-type versus N-type.Trends Pharmacol Sci 2004,25:465-470.
[8]Snutch TP.Targeting chronic and neuropathic pain:the N-type calcium channel comes of age.NeuroRx 2005,2:662-670.
[9]Terlau H,Olivera BM.ConusVenoms:a rich source of novel ion channeltargeted peptides.Physiol Rev,2004,84:41～68.
[10]Kim C,Jun K,Lee T,et al.Altered nociceptive response in mice deficient in the α 1_B subunit of the voltage-dependent calcium channel.Mol Cell Neurosci,2001,18:235～245.
[11]Ino M,Yoshinaga T,Wakamori M,et al.Functional disorders of the sympathetic nervous system in mice lacking thealBsubunit(CaV2.2)of N-type calcium channels.Proc Natl Acad Sci USA,2001,98:5323～5328.
[12]Beuckmann CT,Sinton CM,Miyamoto N,et al.N-type calcium channelαlBsubunit(CaV2.2) knock-out mice display hyperactivity and vigilance state differences.J Neurosci,2003,23:6793-6797.
[13]Saegusa H,Kurihara T,Zong S,et al.Suppression of inflammatory and neuropathic pain symptoms in mice lacking the N-type Ca~(2+)channel.EMBO J,2001,20:2349-2356.
[14]Beuckmann CT,Sinton CM,Miyamoto N,et al.N-type calcium channel α1_β subunit(CaV2.2) knock-out mice display hyperactivity and vigilance state differences.J Neurosci,2003.23:6793-6797.
[15]王红,周晓巍,刘凤云等.神经痛大鼠痛行为与ω-芋螺毒素SO3的影响.中国临床康复,2005,9(14):104-106.
[16]Bell TJ,Thaler C,Castiglioni AJ,et al.Cell-specific alternative splicing increases calcium channel current density in the pain pathway.Neuron,2004,41:127-138.
[17]Zamponi GW,McCleskey EW.Splicing it up:a variant of the N-type calcium channel specific for pain.Neuron,2004,41:3-4.
[18]Castiglioni AJ,Raingo J,Lipscombe D.Alternative splicing in the C- terminus of CaV2.2 controls expression and gating of N-type calcium channels.J Physiol(Lond) 2006,576:119 -134.
[19]Christophe A,Camila S.D,Alexandra E.K et.al.Differential Role of N-Type Calcium Channel Splice Isoforms in Pain.Neuroscience.2007,27(24):6363-6373.
[20]Jesica R,Andrew J C & Diane L.Alternative splicing controls G protein-dependent inhibition of N-type calcium channels in nociceptors.Nature neuroscience.2007,10:285-292
[21]Koyano S,Suzutani T,Yoshida Iet al.Analysis of phosphorylation pathways of antiherpesvirus nucleosides by varicella-zoster virus-specific enzymes[J].AntimicrobAgentsChemother,1996,40(4):920-924.
[22]E.Bourinet,T.W.Soong,K.Sutton,et al.Splicing of alpha 1A subunit gene generates phenotypic variants of P- and Q-type calcium channels.Nat.Neurosci. 1999(2):407-415.
[23]A.M.Beedle,J.E.McRory,O.Poirot,et al.Agonistindependent modulation of N-type calcium channels by ORL1 receptors,Nat.Neurosci.2004,(7):118-125.
[24]A.Maximov,I.Bezprozvanny,Synaptic targeting of N-type calcium channels in hippocampal neurons,J.Neurosci.2002,22:6939-6952.
[25]A.A.Simen,C.C.Lee,B.B.Simen,et al.The C terminus of the Ca channel alpha IB subunit mediates selective inhibition by G-protein-coupled receptors.J.Neurosci.2001,21:7587-7597.
[26]A.Maximov,T.C.Sudhof,I.Bezprozvanny,Association of neuronal calcium channels with modular adaptor proteins,J.Biol.Chem.1999,274:24453-24456.
[27]H.Hibino,R.Pironkova,O.Onwumere,et al.RIM binding proteins(RBPs)couple Rab3-interacting molecules(RIMs)to voltage-gated Ca channels,Neuron 2002,34:411-423.
[28]H.S.Krovetz,T.D.Helton,A.L.Crews,et al.C-Terminal alternative splicing changes the gating properties of a human spinal cord calcium channel alpha 1A subunit,J.Neurosci.2000,20:7564-7570.
[29]H.Liang,CD.DeMaria,M.G.Erickson,et al.Unified mechanisms of Ca~(2+)regulation across the Ca~(2+)channel family,Neuron2003,39:951-960.
[30]Mergulhao F J,Kelly A GMonteiro G A,et al.Troubleshooting in gene splicing by overlap extensions step-wise method.Mol Biotechhol.1999,12(3):285-287.
[31]Knight SW,Bass BL.A role for the RNase Ⅲ enzyme DCR-1 in RNA interference and germ line development in Caenorhabditis elegans.Science.2001,293(5538):2269-2271.
[32]Fire A,Xu S,Montgomery MK,Kostas SA,et alPotent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans.Nature.1998.391(6669):806-811.
[33]Lipardi C,Wei Q,Paterson BM.RNAi as random degradative PCR.siRNA primers convert mRNA into dsRNA that are degraded to generate new siRNAs.Cell,2001,107:297-307.
[34]Elbashir SM,Lendeckel W,Tuschl T.RNA interference is mediated by 21-and 22-nucleotide RNAs.Genes Dev,2001.15(2):188-200.
[35]Brummelkamp TR,Bernards R.Agami R.Stable suppression of tumorigeni city by virus-mediated RNA interference.Cancer Cell.2002,2(3):243-247.
[36]Elbashir SM,Harborth J,Lendeckel W,et al.Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cell.Nature.2001,411(6836):494-498.
[37]Lin SL,Chuong CM,Ying SY.A novel mRNA-cDNA interference phenomenon for silencing bcl-2 expression in human LNCaP cells.Biochem Biophys Res Commun.2001,281(3):639-644.
[38]Scherr M,Morgan MA,Eder M.Gene silencing mediated by small interfering RNAs in mammalian cells.Curr Med Chem.2003,10(3):245-256.
[39]Amarzguioui M,Rossi JJ,Kim D.Approaches for chemically synthesized siRNA and vector-mediated RNAi.FEBS Lett.2005,579(26):5974-5981.
[40]Porreca F,Lai J,Bian D,et al.A comparison of the potential role of the tetrodotoxin-insensitive sodium channels,PN3/SNS and NaN/SNS2,in rat models of chronic pain.Proc Natl Acad Sci USA,1999,96(14):7640-7644.
[41]Sweitzera SM,Petersa MC,Mab JY,et al.Peripheral and central p38MAPK mediates capsaicin-induced hyperalgesia.Pain.2004,1(11):278-285.
[42]Bomer c.Apoptosis without Caspases:an inefficient molecular 13 guillotine.Cell Death Differ.1999,6(6):497-502.
[43]Wieseler-Frank J.Maier SF.Watkins LR.Immune-to-brain communication dynamically modulates pain:physiological and pathological consequences.Brain Behav Immun.2005,19(2):104-110.
[44]Inoue K,Tsuda M.The role of microglia and ATP receptors in a mechanism of neuropathic pain.Nippon Yakurigaku Zasshi.2006,127:14—17.
[45]Altier C,Zamponi GW.Opioid,cheating its receptors,exacerbates pain.Nat neurosci.2006 Dec,9(12):1534-40.
[46]Trono D.Lentiviral vectors:turning a deadly foe into a therapeutioc agent.Gene Ther.2000,7:20-23.
[47]Kafri T.van Praag H.Gage FH.et al.Lentiviral vectors:regulated gene expression.Mol Ther.2000,l:516-521.
[1]C.A.Reid,J.M.Bekkers,J.D.Clements.Presynaptic Ca~(2+)channels:a functional patch work.Trends Neurosci.2003,26:683-687.
[2]K.Dunlap,J.I.Luebke,T.J.Turner,Exocytotic Ca~(2+)channels in mammalian central neurons.Trends Neurosci.1995,18:89-98.
[3]T.A.Brosenitsch,D.M.Katz,Physiological patterns of electrical stimulation can induce neuronal gene expression by activating N-type calcium channels.J.Neurosci.2001,21:2571-2579.
[4]O.T.Jones,G.M.Bernstein,E.J.Jones,et al.N-Type calcium channels in the developing rat hippocampus:subunit,complex,and regional expression,J.Neurosci.1997,17:6152-6164.
[5]Woolf CJ,SalterMW.Neuronal plasticity:increasingthe gain inpain.Science,2000,288:1765-1768
[6]N.M.Soldatov,Genomic structure of human L-type Ca~(2+)channel,Genomics 1994,22:77-87.
[7]D.Lipscombe,J.Q.Pan,A.C.Gray,Functional diversity in neuronal voltagegated calcium channels by alternative splicing of Ca(v)alphal,Mol.Neurobiol.2002,26:21-44.
[8]D.Lipscombe,A.J.Castiglioni,Alternative splicing in voltage gated calcium channels,in:S.I.McDonough(Ed.),Calcium Channel Pharmacology,Kluwer Academic/Plenum,2004,pp.369-409.
[9]M.C.Emerick,R.Stein,R.Kunze,et al.Profiling the array of Ca(v)3.1 variants from the human T-type calcium channel gene CACNAIG:alternative structures,developmental expression,and biophysical variations,Proteins 2006,64:320-342.
[10]M.Missler,T.C.Sudhof,Neurexins:three genes and 1001 products,Trends Genet.1998,14:20-26.
[11]D.Schmucker,J.C.Clemens,H.Shu,et al.Drosophila Dscam is an axon guidance receptor exhibiting extraordinary molecular diversity.Cell 2000,101:671-684.
[12]L.Birnbaumer,N.Qin,R.Olcese,et al.Structures and functions of calcium channel beta subunits,Bioenerg.Biomembr.1998,30:357-375.
[13]N.Klugbauer,E.Marais,F.Hofmann,Calcium channel alpha2delta subunits:differential expression,function,and drug binding,J.Bioenerg.Biomembr.2003,35:639-647.
[14]D.Walker,M.De Waard,Subunit interaction sites in voltagedependent Ca2+ channels:role in channel function,Trends Neurosci.1998,21:148-154.
[15]J.Strock,M.A.Diverse-Pierluissi,Ca~(2+)channels as integrators of G protein-mediated signaling in neurons,Mol.Pharmacol.2004,66:1071-1076.
[16]S.R.Ikeda,K.Dunlap,Voltage-dependent modulation of N-type calcium channels:role of G protein subunits,Adv.Second Messenger Phosphoprotein Res.1999,33:131-151.
[17]B.Hille,D.J.Beech,L.Bernheim,et al.Multiple G-protein-coupled pathways inhibit N-type Ca channels of neurons,Life Sci.1995,56:989-992.
[18]J.Striessnig,Targeting voltage-gated Ca~(2+)channels,Lancet.2001,357:1294.
[19]A.Maximov,I.Bezprozvanny,Synaptic targeting of N-type calcium channels in hippocampal neurons,Neurosci.2002,22:6939-6952.
[20]Pan,J.Q.& Lipscombe,D.Neurosci.2000,20:4769-4775.
[21]E.Bourinet,T.W.Soong,K.Sutton,et al.Splicing of alpha 1A subunit gene generates phenotypic variants of P-and Q-type calcium channels,Nat.Neurosci.1999,2:407-415.
[22]A.M.Beedle,J.E.McRory,O.Poirot,et al.Agonistindependent modulation of N-type calcium channels by ORL1 receptors,Nat.Neurosci.2004,7:118-125.
[23]A.A.Simen,C.C.Lee,B.B.Simen,et al.The C terminus of the Ca channel alphalB subunit mediates selective inhibition by G-protein-coupled receptors, Neurosci.2001,21:7587-7597.
[24]A.Maximov,T.C.Sudhof,I.Bezprozvanny,Association of neuronal calcium channels with modular adaptor proteins,Biol.Chem.1999,274:24453-24456.
[25]H.Hibino,R.Pironkova,O.Onwumere,et al.RIM binding proteins(RBPs)couple Rab3-interacting molecules(RIMs)to voltage-gated Ca(2+)channels,Neuron 2002,34:411-423.
[26]H.S.Krovetz,T.D.Helton,A.L.Crews,et al.C-Terminal alternative splicing changes the gating properties of a human spinal cord calcium channel alpha 1A subunit,Neurosci.2000,20:7564-7570.
[27]H.Liang,CD.DeMaria,M.G.Erickson,et al.Unified mechanisms of Ca2+ regulation across the Ca2+ channel family,Neuron2003,39:951-960.
[28]L.M.Kerr,F.Filloux,B.M.Olivera,et al.Autoradiographic localization of calcium channels with[125I]omegaconotoxin in rat brain,Eur.J.Pharmacol.1988,146:181-183.
[29]C.A.Maggi,M.Tramontana,R.Cecconi,et al.Neurochemical evidence for the involvement of N-type calcium channels in transmitter secretion from peripheral endings of sensory nerves in guinea pigs,Neurosci.Lett.1990,114:203-206.
[30]Holz GGt,K.Dunlap,R.M.Kream,Characterization of the electrically evoked release of substance P from dorsal root ganglion neurons:methods and dihydropyridine sensitivity,J.Neurosci.1988,8:463-471.
[31]B.Heinke,E.Balzer,J.Sandkuhler,Pre-and postsynaptic contributions of voltage-dependent Ca2+ channels to nociceptive transmission in rat spinal lamina I neurons,Eur.J.Neurosci.2004,19:103-111.
[32]Bell TJ,Thaler C,Castiglioni AJ,et al.Cell-specific alternative splicing increases calcium channel current density in the pain pathway.Neuron.2004,41:127～138.
[33]Zamponi GW,McCleskey EW.Splicing it up:a variant of the N-type calcium channel specific for pain.Neuron,2004,41:3～4.
[34]Andrew J.Castiglioni,Jesica Raingoet al.Alternative splicing in the C-terminus of CaV2.2 controls expression and gating of N-type calcium channels.Physiol 2006,576.1:119-134
[35]K.Dunlap,G.D.Fischbach,Neurotransmitters decrease the calcium ocmponent of sensory neurone action potentials,Nature1978,276:837-839.
[36]Roche,K.W.et al.Molecular determinants of NMDA receptor internalization.Nat.Neurosci.2001,4:794-802.
[37]D.Lipscombe,S.Kongsamut,R.W.Tsien,Alpha-adrenergic inhibition of sympathetic neurotransmitter release mediated by modulation of N-type calcium-channel gating,Nature 1989,340:639-642.
[38]Ikeda,S.R.& Dunlap,K.Voltage-dependent modulation of N-type calcium channels:role of G protein subunits.Adv.Second Messenger Phosphoprotein Res.1999,33:131-151.
[39]Diverse-Pierluissi,M.& Dunlap,K.Distinct,convergent second messenger pathways modulate neuronal calcium currents.Neuron 1993,10:753-760.
[40]K.S.Elmslie,Neurotransmitter modulation of neuronal calcium channels,J.Bioenerg.Biomembr.200335:477-489.
[41]D.L.Brody,P.G.Patil,J.G.Mulle,et al.Bursts of action potential waveforms relieve G-protein inhibition of recombinant P/Q-type Ca2+ channels in HEK 293 cells,J.Physiol.1997499(3):637-644.
[42]M.Diverse-Pierluissi,K.Dunlap,Distinct,convergent second messenger pathways modulate neuronal calcium currents,Neuron 1993,10:753-760.
[43]S.G.Rane,K.Dunlap,Kinase C activator 1.2-oleoylacetylglycerol attenuates voltage-dependent calcium current in sensory neurons,Proc.Natl.Acad.Sci.U S A 1986,83:184-188.
[44]Jesica R,Andrew J C.Diane L.Alternative splicing controls G protein-dependent inhibition of N-type calcium channels in nociceptors.Nature neuroscience,2007,10:285-292
[45]Altier,C.et al.0RL1 receptor-mediated internalization of N-type calcium channels.Nat.Neurosci.2006,9:31-40.
[46]Tombler,E.G protein-induced trafficking of voltage-dependent calcium channels.J.Biol.Chem.2006,281:1827-1839.
[47]Lipscombe,D.Raingo,J.Internalizing channels:a mechanism to control pain? Nat.Neurosci.2006,9:8-10(2006).
[48]Bonifacino,J.S.Traub,L.M.Signals for sorting of transmembrane proteins to endosomes and lysosomes.Annu.Rev.Biochem.2003,72:395-447.
[49]Christophe A,Camila S.D,Alexandra E.K et.al.Differential Role of N-Type Calcium Channel Splice Isoforms in Pain.Neuroscience.2007,27(24):63 63-6373.
[2]K.Dunlap,J.I.Luebke.T.J.Turner.Exocytotic Ca channels in mammalian central neurons.Trends Neurosci.1995,18:89-98.
[3]Jarvis ES,Zamponi GW.Interactions between presynaptic Ca2+channels,cytoplasmic messengers and proteins of the synaptic vesicle release complex.Trends Pharmacol Sci,2001,22:519-525.
[4]Augustine G.How does calcium trigger neurotransmitter release?Curr Opin Neurobiol,2001,11:320-326.
[5]T.A.Brosenitsch,D.M.Katz,Physiological patterns of electrical stimulation can induce neuronal gene expression by activating N-type calcium channels,J.Neurosci.2001,21:2571-2579.
[6]O.T.Jones,G.M.Bernstein,E.J.Jones,et al.N-Type calcium channels in the developing rat hippocampus:subunit,complex,and regional expression,J.Neurosci.1997,17:6152-6164.
[7]Altier C,Zamponi GW.Targeting Ca~(2+)channels to treat pain:T-type versus N-type.Trends Pharmacol Sci 2004,25:465-470.
[8]Snutch TP.Targeting chronic and neuropathic pain:the N-type calcium channel comes of age.NeuroRx 2005,2:662-670.
[9]Terlau H,Olivera BM.ConusVenoms:a rich source of novel ion channeltargeted peptides.Physiol Rev,2004,84:41～68.
[10]Kim C,Jun K,Lee T,et al.Altered nociceptive response in mice deficient in the α 1_B subunit of the voltage-dependent calcium channel.Mol Cell Neurosci,2001,18:235～245.
[11]Ino M,Yoshinaga T,Wakamori M,et al.Functional disorders of the sympathetic nervous system in mice lacking thealBsubunit(CaV2.2)of N-type calcium channels.Proc Natl Acad Sci USA,2001,98:5323～5328.
[12]Beuckmann CT,Sinton CM,Miyamoto N,et al.N-type calcium channelαlBsubunit(CaV2.2) knock-out mice display hyperactivity and vigilance state differences.J Neurosci,2003,23:6793-6797.
[13]Saegusa H,Kurihara T,Zong S,et al.Suppression of inflammatory and neuropathic pain symptoms in mice lacking the N-type Ca~(2+)channel.EMBO J,2001,20:2349-2356.
[14]Beuckmann CT,Sinton CM,Miyamoto N,et al.N-type calcium channel α1_β subunit(CaV2.2) knock-out mice display hyperactivity and vigilance state differences.J Neurosci,2003.23:6793-6797.
[15]王红,周晓巍,刘凤云等.神经痛大鼠痛行为与ω-芋螺毒素SO3的影响.中国临床康复,2005,9(14):104-106.
[16]Bell TJ,Thaler C,Castiglioni AJ,et al.Cell-specific alternative splicing increases calcium channel current density in the pain pathway.Neuron,2004,41:127-138.
[17]Zamponi GW,McCleskey EW.Splicing it up:a variant of the N-type calcium channel specific for pain.Neuron,2004,41:3-4.
[18]Castiglioni AJ,Raingo J,Lipscombe D.Alternative splicing in the C- terminus of CaV2.2 controls expression and gating of N-type calcium channels.J Physiol(Lond) 2006,576:119 -134.
[19]Christophe A,Camila S.D,Alexandra E.K et.al.Differential Role of N-Type Calcium Channel Splice Isoforms in Pain.Neuroscience.2007,27(24):6363-6373.
[20]Jesica R,Andrew J C & Diane L.Alternative splicing controls G protein-dependent inhibition of N-type calcium channels in nociceptors.Nature neuroscience.2007,10:285-292
[21]Koyano S,Suzutani T,Yoshida Iet al.Analysis of phosphorylation pathways of antiherpesvirus nucleosides by varicella-zoster virus-specific enzymes[J].AntimicrobAgentsChemother,1996,40(4):920-924.
[22]E.Bourinet,T.W.Soong,K.Sutton,et al.Splicing of alpha 1A subunit gene generates phenotypic variants of P- and Q-type calcium channels.Nat.Neurosci. 1999(2):407-415.
[23]A.M.Beedle,J.E.McRory,O.Poirot,et al.Agonistindependent modulation of N-type calcium channels by ORL1 receptors,Nat.Neurosci.2004,(7):118-125.
[24]A.Maximov,I.Bezprozvanny,Synaptic targeting of N-type calcium channels in hippocampal neurons,J.Neurosci.2002,22:6939-6952.
[25]A.A.Simen,C.C.Lee,B.B.Simen,et al.The C terminus of the Ca channel alpha IB subunit mediates selective inhibition by G-protein-coupled receptors.J.Neurosci.2001,21:7587-7597.
[26]A.Maximov,T.C.Sudhof,I.Bezprozvanny,Association of neuronal calcium channels with modular adaptor proteins,J.Biol.Chem.1999,274:24453-24456.
[27]H.Hibino,R.Pironkova,O.Onwumere,et al.RIM binding proteins(RBPs)couple Rab3-interacting molecules(RIMs)to voltage-gated Ca channels,Neuron 2002,34:411-423.
[28]H.S.Krovetz,T.D.Helton,A.L.Crews,et al.C-Terminal alternative splicing changes the gating properties of a human spinal cord calcium channel alpha 1A subunit,J.Neurosci.2000,20:7564-7570.
[29]H.Liang,CD.DeMaria,M.G.Erickson,et al.Unified mechanisms of Ca~(2+)regulation across the Ca~(2+)channel family,Neuron2003,39:951-960.
[30]Mergulhao F J,Kelly A GMonteiro G A,et al.Troubleshooting in gene splicing by overlap extensions step-wise method.Mol Biotechhol.1999,12(3):285-287.
[31]Knight SW,Bass BL.A role for the RNase Ⅲ enzyme DCR-1 in RNA interference and germ line development in Caenorhabditis elegans.Science.2001,293(5538):2269-2271.
[32]Fire A,Xu S,Montgomery MK,Kostas SA,et alPotent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans.Nature.1998.391(6669):806-811.
[33]Lipardi C,Wei Q,Paterson BM.RNAi as random degradative PCR.siRNA primers convert mRNA into dsRNA that are degraded to generate new siRNAs.Cell,2001,107:297-307.
[34]Elbashir SM,Lendeckel W,Tuschl T.RNA interference is mediated by 21-and 22-nucleotide RNAs.Genes Dev,2001.15(2):188-200.
[35]Brummelkamp TR,Bernards R.Agami R.Stable suppression of tumorigeni city by virus-mediated RNA interference.Cancer Cell.2002,2(3):243-247.
[36]Elbashir SM,Harborth J,Lendeckel W,et al.Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cell.Nature.2001,411(6836):494-498.
[37]Lin SL,Chuong CM,Ying SY.A novel mRNA-cDNA interference phenomenon for silencing bcl-2 expression in human LNCaP cells.Biochem Biophys Res Commun.2001,281(3):639-644.
[38]Scherr M,Morgan MA,Eder M.Gene silencing mediated by small interfering RNAs in mammalian cells.Curr Med Chem.2003,10(3):245-256.
[39]Amarzguioui M,Rossi JJ,Kim D.Approaches for chemically synthesized siRNA and vector-mediated RNAi.FEBS Lett.2005,579(26):5974-5981.
[40]Porreca F,Lai J,Bian D,et al.A comparison of the potential role of the tetrodotoxin-insensitive sodium channels,PN3/SNS and NaN/SNS2,in rat models of chronic pain.Proc Natl Acad Sci USA,1999,96(14):7640-7644.
[41]Sweitzera SM,Petersa MC,Mab JY,et al.Peripheral and central p38MAPK mediates capsaicin-induced hyperalgesia.Pain.2004,1(11):278-285.
[42]Bomer c.Apoptosis without Caspases:an inefficient molecular 13 guillotine.Cell Death Differ.1999,6(6):497-502.
[43]Wieseler-Frank J.Maier SF.Watkins LR.Immune-to-brain communication dynamically modulates pain:physiological and pathological consequences.Brain Behav Immun.2005,19(2):104-110.
[44]Inoue K,Tsuda M.The role of microglia and ATP receptors in a mechanism of neuropathic pain.Nippon Yakurigaku Zasshi.2006,127:14—17.
[45]Altier C,Zamponi GW.Opioid,cheating its receptors,exacerbates pain.Nat neurosci.2006 Dec,9(12):1534-40.
[46]Trono D.Lentiviral vectors:turning a deadly foe into a therapeutioc agent.Gene Ther.2000,7:20-23.
[47]Kafri T.van Praag H.Gage FH.et al.Lentiviral vectors:regulated gene expression.Mol Ther.2000,l:516-521.
[1]C.A.Reid,J.M.Bekkers,J.D.Clements.Presynaptic Ca~(2+)channels:a functional patch work.Trends Neurosci.2003,26:683-687.
[2]K.Dunlap,J.I.Luebke,T.J.Turner,Exocytotic Ca~(2+)channels in mammalian central neurons.Trends Neurosci.1995,18:89-98.
[3]T.A.Brosenitsch,D.M.Katz,Physiological patterns of electrical stimulation can induce neuronal gene expression by activating N-type calcium channels.J.Neurosci.2001,21:2571-2579.
[4]O.T.Jones,G.M.Bernstein,E.J.Jones,et al.N-Type calcium channels in the developing rat hippocampus:subunit,complex,and regional expression,J.Neurosci.1997,17:6152-6164.
[5]Woolf CJ,SalterMW.Neuronal plasticity:increasingthe gain inpain.Science,2000,288:1765-1768
[6]N.M.Soldatov,Genomic structure of human L-type Ca~(2+)channel,Genomics 1994,22:77-87.
[7]D.Lipscombe,J.Q.Pan,A.C.Gray,Functional diversity in neuronal voltagegated calcium channels by alternative splicing of Ca(v)alphal,Mol.Neurobiol.2002,26:21-44.
[8]D.Lipscombe,A.J.Castiglioni,Alternative splicing in voltage gated calcium channels,in:S.I.McDonough(Ed.),Calcium Channel Pharmacology,Kluwer Academic/Plenum,2004,pp.369-409.
[9]M.C.Emerick,R.Stein,R.Kunze,et al.Profiling the array of Ca(v)3.1 variants from the human T-type calcium channel gene CACNAIG:alternative structures,developmental expression,and biophysical variations,Proteins 2006,64:320-342.
[10]M.Missler,T.C.Sudhof,Neurexins:three genes and 1001 products,Trends Genet.1998,14:20-26.
[11]D.Schmucker,J.C.Clemens,H.Shu,et al.Drosophila Dscam is an axon guidance receptor exhibiting extraordinary molecular diversity.Cell 2000,101:671-684.
[12]L.Birnbaumer,N.Qin,R.Olcese,et al.Structures and functions of calcium channel beta subunits,Bioenerg.Biomembr.1998,30:357-375.
[13]N.Klugbauer,E.Marais,F.Hofmann,Calcium channel alpha2delta subunits:differential expression,function,and drug binding,J.Bioenerg.Biomembr.2003,35:639-647.
[14]D.Walker,M.De Waard,Subunit interaction sites in voltagedependent Ca2+ channels:role in channel function,Trends Neurosci.1998,21:148-154.
[15]J.Strock,M.A.Diverse-Pierluissi,Ca~(2+)channels as integrators of G protein-mediated signaling in neurons,Mol.Pharmacol.2004,66:1071-1076.
[16]S.R.Ikeda,K.Dunlap,Voltage-dependent modulation of N-type calcium channels:role of G protein subunits,Adv.Second Messenger Phosphoprotein Res.1999,33:131-151.
[17]B.Hille,D.J.Beech,L.Bernheim,et al.Multiple G-protein-coupled pathways inhibit N-type Ca channels of neurons,Life Sci.1995,56:989-992.
[18]J.Striessnig,Targeting voltage-gated Ca~(2+)channels,Lancet.2001,357:1294.
[19]A.Maximov,I.Bezprozvanny,Synaptic targeting of N-type calcium channels in hippocampal neurons,Neurosci.2002,22:6939-6952.
[20]Pan,J.Q.& Lipscombe,D.Neurosci.2000,20:4769-4775.
[21]E.Bourinet,T.W.Soong,K.Sutton,et al.Splicing of alpha 1A subunit gene generates phenotypic variants of P-and Q-type calcium channels,Nat.Neurosci.1999,2:407-415.
[22]A.M.Beedle,J.E.McRory,O.Poirot,et al.Agonistindependent modulation of N-type calcium channels by ORL1 receptors,Nat.Neurosci.2004,7:118-125.
[23]A.A.Simen,C.C.Lee,B.B.Simen,et al.The C terminus of the Ca channel alphalB subunit mediates selective inhibition by G-protein-coupled receptors, Neurosci.2001,21:7587-7597.
[24]A.Maximov,T.C.Sudhof,I.Bezprozvanny,Association of neuronal calcium channels with modular adaptor proteins,Biol.Chem.1999,274:24453-24456.
[25]H.Hibino,R.Pironkova,O.Onwumere,et al.RIM binding proteins(RBPs)couple Rab3-interacting molecules(RIMs)to voltage-gated Ca(2+)channels,Neuron 2002,34:411-423.
[26]H.S.Krovetz,T.D.Helton,A.L.Crews,et al.C-Terminal alternative splicing changes the gating properties of a human spinal cord calcium channel alpha 1A subunit,Neurosci.2000,20:7564-7570.
[27]H.Liang,CD.DeMaria,M.G.Erickson,et al.Unified mechanisms of Ca2+ regulation across the Ca2+ channel family,Neuron2003,39:951-960.
[28]L.M.Kerr,F.Filloux,B.M.Olivera,et al.Autoradiographic localization of calcium channels with[125I]omegaconotoxin in rat brain,Eur.J.Pharmacol.1988,146:181-183.
[29]C.A.Maggi,M.Tramontana,R.Cecconi,et al.Neurochemical evidence for the involvement of N-type calcium channels in transmitter secretion from peripheral endings of sensory nerves in guinea pigs,Neurosci.Lett.1990,114:203-206.
[30]Holz GGt,K.Dunlap,R.M.Kream,Characterization of the electrically evoked release of substance P from dorsal root ganglion neurons:methods and dihydropyridine sensitivity,J.Neurosci.1988,8:463-471.
[31]B.Heinke,E.Balzer,J.Sandkuhler,Pre-and postsynaptic contributions of voltage-dependent Ca2+ channels to nociceptive transmission in rat spinal lamina I neurons,Eur.J.Neurosci.2004,19:103-111.
[32]Bell TJ,Thaler C,Castiglioni AJ,et al.Cell-specific alternative splicing increases calcium channel current density in the pain pathway.Neuron.2004,41:127～138.
[33]Zamponi GW,McCleskey EW.Splicing it up:a variant of the N-type calcium channel specific for pain.Neuron,2004,41:3～4.
[34]Andrew J.Castiglioni,Jesica Raingoet al.Alternative splicing in the C-terminus of CaV2.2 controls expression and gating of N-type calcium channels.Physiol 2006,576.1:119-134
[35]K.Dunlap,G.D.Fischbach,Neurotransmitters decrease the calcium ocmponent of sensory neurone action potentials,Nature1978,276:837-839.
[36]Roche,K.W.et al.Molecular determinants of NMDA receptor internalization.Nat.Neurosci.2001,4:794-802.
[37]D.Lipscombe,S.Kongsamut,R.W.Tsien,Alpha-adrenergic inhibition of sympathetic neurotransmitter release mediated by modulation of N-type calcium-channel gating,Nature 1989,340:639-642.
[38]Ikeda,S.R.& Dunlap,K.Voltage-dependent modulation of N-type calcium channels:role of G protein subunits.Adv.Second Messenger Phosphoprotein Res.1999,33:131-151.
[39]Diverse-Pierluissi,M.& Dunlap,K.Distinct,convergent second messenger pathways modulate neuronal calcium currents.Neuron 1993,10:753-760.
[40]K.S.Elmslie,Neurotransmitter modulation of neuronal calcium channels,J.Bioenerg.Biomembr.200335:477-489.
[41]D.L.Brody,P.G.Patil,J.G.Mulle,et al.Bursts of action potential waveforms relieve G-protein inhibition of recombinant P/Q-type Ca2+ channels in HEK 293 cells,J.Physiol.1997499(3):637-644.
[42]M.Diverse-Pierluissi,K.Dunlap,Distinct,convergent second messenger pathways modulate neuronal calcium currents,Neuron 1993,10:753-760.
[43]S.G.Rane,K.Dunlap,Kinase C activator 1.2-oleoylacetylglycerol attenuates voltage-dependent calcium current in sensory neurons,Proc.Natl.Acad.Sci.U S A 1986,83:184-188.
[44]Jesica R,Andrew J C.Diane L.Alternative splicing controls G protein-dependent inhibition of N-type calcium channels in nociceptors.Nature neuroscience,2007,10:285-292
[45]Altier,C.et al.0RL1 receptor-mediated internalization of N-type calcium channels.Nat.Neurosci.2006,9:31-40.
[46]Tombler,E.G protein-induced trafficking of voltage-dependent calcium channels.J.Biol.Chem.2006,281:1827-1839.
[47]Lipscombe,D.Raingo,J.Internalizing channels:a mechanism to control pain? Nat.Neurosci.2006,9:8-10(2006).
[48]Bonifacino,J.S.Traub,L.M.Signals for sorting of transmembrane proteins to endosomes and lysosomes.Annu.Rev.Biochem.2003,72:395-447.
[49]Christophe A,Camila S.D,Alexandra E.K et.al.Differential Role of N-Type Calcium Channel Splice Isoforms in Pain.Neuroscience.2007,27(24):63 63-6373.