Identification of peptide components of the brevetoxin receptor site of rat brain sodium channels.
Identification of peptide components of the brevetoxin receptor site of rat brain sodium channels.
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发表时间：
1994-08
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通讯作者：
V. Trainer;D. Baden;W. Catterall
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作者：
V. Trainer;D. Baden;W. Catterall
为了鉴定作用于Na+通道受体5位点的脂溶性神经毒素brevetoxins的结合域，用对叠氮苯甲酰氚标记的brevetoxin对纯化和重组的大鼠脑Na+通道进行了光标记，并对标记肽进行了鉴定。利用凝胶切片和荧光技术发现了一个与Na+通道α -亚基对应的表观分子质量为250 kDa的放射性标记带。由该配体特异性光标记的α -亚基区域然后通过蛋白水解片段的抗体定位来鉴定。即使经过广泛的蛋白水解，识别Na+通道跨膜段IS6和IVS5内或邻近氨基酸序列的抗肽抗体也能免疫沉淀高达40%的标记肽。将短草毒素光标记物掺入洗涤液中纯化Na+通道的α -亚基中，获得了更广泛的胰蛋白酶消化。特异性免疫沉淀的6-kDa肽含有跨膜片段S6，如果胰蛋白酶消化完全，则标记的肽片段限制在Thr-400至Lys-443残基上，如果胰蛋白酶切割不完全，则标记的肽片段限制在Ala-396至Lys-455残基上。同样，从IV结构域鉴定特异性免疫沉淀的6-kDa肽，将标记的肽限制在跨膜段S5细胞外侧的Glu-1738至Lys-1785或Glu-1738至Lys-1793残基上。这些结果为跨膜片段IS6和IVS5在Na+通道α亚基的天然构象中密切相关提供了直接证据，并暗示它们的相互作用区域是短草毒素受体位点的重要组成部分。
To identify the binding domain for brevetoxins, a family of lipid-soluble neurotoxins acting at Na+ channel receptor site 5, purified and reconstituted rat brain Na+ channels were photolabeled with p-azidobenzoyl tritium-labeled brevetoxin, and the labeled peptides were identified. A radiolabeled band with an apparent molecular mass of 250 kDa corresponding to the Na+ channel alpha-subunit was revealed using both gel slicing and fluorography techniques. Regions of the alpha-subunit specifically photolabeled by this ligand were then identified by antibody mapping of proteolytic fragments. Even after extensive proteolysis, anti-peptide antibodies recognizing amino acid sequences within or adjacent to Na+ channel transmembrane segments IS6 and IVS5 were each able to immunoprecipitate up to 40% of the labeled peptides. A more extensive tryptic digest was obtained with a preparation in which the brevetoxin photolabel was incorporated into the alpha-subunit of purified Na+ channel in detergent solution. The identification of a specifically immunoprecipitated 6-kDa peptide containing transmembrane segment S6 from domain I restricted the labeled peptide fragment to residues Thr-400 to Lys-443 if tryptic digestion was complete or Ala-396 to Lys-455 if tryptic cleavage was incomplete. Similarly, the identification of a specifically immunoprecipitated 6-kDa peptide from domain IV restricted the labeled peptide to residues Glu-1738 to Lys-1785 or Glu-1738 to Lys-1793 on the extracellular side of transmembrane segment S5. These results provide direct evidence for close association of transmembrane segments IS6 and IVS5 in the native conformation of the Na+ channel alpha-subunit and implicate their region of interaction as an important component of the brevetoxin receptor site.