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Abstract：
Genetic and functional studies have confirmed an important role for the voltage-gated sodium channel Nav1.9 in human pain disorders. However, low functional expression of Nav1.9 in heterologous systems (e.g. in human embryonic kidney 293 (HEK293) cells) has hampered studies of its biophysical and pharmacological properties and the development of high-throughput assays for drug development targeting this channel. The mechanistic basis for the low level of Nav1.9 currents in heterologous expression systems is not understood. Here, we implemented a multidisciplinary approach to investigate the mechanisms that govern functional Nav1.9 expression. Recombinant expression of a series of Nav1.9-Nav1.7 C-terminal chimeras in HEK293 cells identified a 49-amino-acid-long motif in the C terminus of the two channels that regulates expression levels of these chimeras. We confirmed the critical role of this motif in the context of a full-length channel chimera, Nav1.9-Ct49aaNav1.7, which displayed significantly increased current density in HEK293 cells while largely retaining the characteristic Nav1.9-gating properties. High-resolution live microscopy indicated that the newly identified C-terminal motif dramatically increases the number of channels on the plasma membrane of HEK293 cells. Molecular modeling results suggested that this motif is exposed on the cytoplasmic face of the folded C terminus, where it might interact with other channel partners. These findings reveal that a 49-residue-long motif in Nav1.9 regulates channel trafficking to the plasma membrane.
摘要：
遗传和功能研究已经证实电压门控钠通道Nav1.9在人类疼痛障碍中的重要作用。然而,Nav1.9在异源系统中(例如在人胚肾293(HEK293)细胞中)的低功能表达阻碍了对其生物物理和药理学性质的研究以及靶向该通道的药物开发的高通量测定的开发。不了解异源表达系统中低水平的Nav1.9电流的机理基础。这里,我们实施了多学科方法来研究控制功能Nav1.9表达的机制.一系列Nav1.9-Nav1.7C端嵌合体在HEK293细胞中的重组表达鉴定了两个通道的C端中的49个氨基酸长的基序,其调节这些嵌合体的表达水平。我们证实了该基序在全长通道嵌合体中的关键作用，Nav1.9-Ct49aNav1.7,其在HEK293细胞中显示出显著增加的电流密度,同时在很大程度上保留了特征Nav1.9门控性质。高分辨率实时显微镜检查表明，新鉴定的C末端基序显着增加了HEK293细胞质膜上的通道数量。分子建模结果表明，该基序暴露在折叠的C末端的细胞质面上，它可能与其他渠道合作伙伴进行交互。这些发现表明，Nav1.9中49个残基长的基序调节向质膜的通道运输。
