TRIP8b regulates HCN1 channel trafficking and gating through two distinct C-terminal interaction sites.
TRIP8b regulates HCN1 channel trafficking and gating through two distinct C-terminal interaction sites.
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DOI：
10.1523/jneurosci.5707-10.2011
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发表时间：
2011-03-16
期刊：
影响因子：
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通讯作者：
Siegelbaum SA
中科院分区：
文献类型：
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作者：
Santoro B;Hu L;Liu H;Saponaro A;Pian P;Piskorowski RA;Moroni A;Siegelbaum SA
脑中超极化激活的环核苷酸调节（HCN）通道与其辅助亚基TRIP 8b（也称为PEX 5 R）相关，TRIP 8b是一种表达为选择性剪接同种型家族的细胞质蛋白。最近的体外和体内研究表明，TRIP 8b与HCN亚基的结合既抑制通道开放又改变通道膜运输，其中一些剪接变体增加通道表面表达，另一些剪接变体降低通道表面表达。在这里，我们解决了小鼠TRIP 8b和HCN 1之间的相互作用的监管结构基础。我们发现，HCN 1和TRIP 8b相互作用在两个不同的网站：一个上游网站，其中的C-接头/环核苷酸结合域的HCN 1相互作用的80个氨基酸的结构域在TRIP 8b的保守的中央核心，和下游网站，其中的C-末端SNL三肽的通道与TRIP 8b的tetratricopeptide重复结构域相互作用。这两个相互作用位点在TRIP 8b对HCN 1运输和门控的影响中发挥不同的功能作用。上游位点的结合是TRIP 8b抑制通道开放的必要条件和充分条件。它也足以介导那些下调通道表面表达的TRIP 8b同种型与TRIP 8b的N-末端区域中存在的运输基序组合的运输效应。相反，在下游相互作用位点的结合用于稳定TRIP 8b的C-末端结构域，允许HCN 1和TRIP 8b之间的最佳相互作用以及介导TRIP 8b对HCN 1通道运输的作用的分子复合物的适当组装。
Hyperpolarization-activated cyclic nucleotide-regulated (HCN) channels in the brain associate with their auxiliary subunit TRIP8b (also known as PEX5R), a cytoplasmic protein expressed as a family of alternatively spliced isoforms. Recent in vitro and in vivo studies have shown that association of TRIP8b with HCN subunits both inhibits channel opening and alters channel membrane trafficking, with some splice variants increasing and others decreasing channel surface expression. Here, we address the structural bases of the regulatory interactions between mouse TRIP8b and HCN1. We find that HCN1 and TRIP8b interact at two distinct sites: an upstream site where the C-linker/cyclic nucleotide-binding domain of HCN1 interacts with an 80 amino acid domain in the conserved central core of TRIP8b, and a downstream site where the C-terminal -SNL tripeptide of the channel interacts with the tetratricopeptide repeat domain of TRIP8b. These two interaction sites play distinct functional roles in the effects of TRIP8b on HCN1 trafficking and gating. Binding at the upstream site is both necessary and sufficient for TRIP8b to inhibit channel opening. It is also sufficient to mediate the trafficking effects of those TRIP8b isoforms that downregulate channel surface expression, in combination with the trafficking motifs present in the N-terminal region of TRIP8b. In contrast, binding at the downstream interaction site serves to stabilize the C-terminal domain of TRIP8b, allowing for optimal interaction between HCN1 and TRIP8b as well as for proper assembly of the molecular complexes that mediate the effects of TRIP8b on HCN1 channel trafficking.