Heterologous expression of NaV1.9 chimeras in various cell systems
Heterologous expression of NaV1.9 chimeras in various cell systems
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DOI：
10.1007/s00424-015-1709-1
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发表时间：
2015-12-01
影响因子：
4.5
通讯作者：
Heinemann, Stefan H.
中科院分区：
文献类型：
--
作者：
Goral, R. Oliver;Leipold, Enrico;Heinemann, Stefan H.
SCN11A 编码电压门控钠通道 Na(V)1.9，它与哺乳动物中表达的其他八个 Na-V 通道的差异最大。它的特点是对原型 Na-V 通道阻断剂河豚毒素具有抗性，并表现出缓慢的激活和失活门控。它在背根神经节神经元中的表达表明其在运动或疼痛信号传导功能中发挥作用，最近人类 SCN11A 中各种突变的发生导致痛觉综合征的改变也证明了这一点。然而，由于宿主细胞中的异源表达非常差，人类 Na(V)1.9 的系统研究受到严重阻碍。使用膜片钳和两电极电压钳方法，我们表明这种限制是由 Na(V)1.9 的 C 端结构引起的。带有 Na(V)1.4 C 末端的 Na(V)1.9 嵌合体不仅在神经元细胞中产生功能性表达，而且在非兴奋性细胞（如 HEK 293T 或非洲爪蟾卵母细胞）中产生功能性表达。嵌合通道相对于 Na(V)1.9 的主要功能差异是加速激活和失活。由于保留了整个跨膜结构域，因此它适合研究通道的药理学特性和致病突变的功能影响。此外，我们还证明了突变S360Y如何使Na(V)1.9通道对河豚毒素和石房蛤毒素敏感，并且Na(V)1.9异常缓慢的开放态失活也是由位于连接结构域III和IV的接头中的IFM（异亮氨酸-苯丙氨酸-蛋氨酸）失活基序介导的。
SCN11A encodes the voltage-gated sodium channel Na(V)1.9, which deviates most strongly from the other eight Na-V channels expressed in mammals. It is characterized by resistance to the prototypic Na-V channel blocker tetrodotoxin and exhibits slow activation and inactivation gating. Its expression in dorsal root ganglia neurons suggests a role in motor or pain signaling functions as also recently demonstrated by the occurrence of various mutations in human SCN11A leading to altered pain sensation syndromes. The systematic investigation of human Na(V)1.9, however, is severely hampered because of very poor heterologous expression in host cells. Using patch-clamp and two-electrode voltage-clamp methods, we show that this limitation is caused by the C-terminal structure of Na(V)1.9. A chimera of Na(V)1.9 harboring the C terminus of Na(V)1.4 yields functional expression not only in neuronal cells but also in non-excitable cells, such as HEK 293T or Xenopus oocytes. The major functional difference of the chimeric channel with respect to Na(V)1.9 is an accelerated activation and inactivation. Since the entire transmembrane domain is preserved, it is suited for studying pharmacological properties of the channel and the functional impact of disease-causing mutations. Moreover, we demonstrate how mutation S360Y makes Na(V)1.9 channels sensitive to tetrodotoxin and saxitoxin and that the unusual slow open-state inactivation of Na(V)1.9 is also mediated by the IFM (isoleucine-phenylalanine-methionine) inactivation motif located in the linker connecting domains III and IV.