ANALYSES OF MOLECULAR MECHANISM OF STRETCH-ACTIVATED CHANNELS USING SINGI

本研究小组鉴定的拉伸激活和Ca（2+）激活的大K通道（SAKcaC）是SIo-1基因编码的Moe通道的一种剪接变体，具有strex序列，一个位于通道细胞质侧的59个氨基添加剪接插入，可能与SAKcaC的膜拉伸敏感性有关。最近，我们发现大多数SAKcaCs在质膜上表现出简单的扩散，但大约25%的旧通道被固定在焦接触面上或靠近焦接触（FC），这表明SAKcaCs与FC组分（如粘附分子和细胞骨架）相关，形成机械传感装置。然而，这种通道固定在FC上的机制尚不清楚。为了解决这个问题，我们研究了STREX在通道固定中的作用。我们表达了gfp标记的STREX，并使用单荧光团视频成像技术进行观察。与rfp标记的paxillin同时观察发现，STREX分子在质膜上被招募并迅速扩散，但其中很大一部分被固定在FC上，这表明STREX本身可以与某些FC成分结合。然而，strex删除的SAKcaCs也固定在fc上或附近，这表明SAKcaCs可能通过STEER和/或通道的其他位点与Its相关联

Stretch activated and Ca (2+) activated big K channel (SAKcaC) identified by our group, is one form of splice variants of the Moe channel encoded by SIo-1 gene, and has the STREX-sequence, a 59 amino-add splice insert located in the cytoplasmic side of the channel, which may responsible fir the membrane stretch sensitivity of SAKcaCs. Recently, we found that most SAKcaCs exhibited simple diffusion on the plasma membrane but that approximatly 25% oldie channels was immobilized on or near focal contacts (FCs), suggesting that SAKcaCs are associated with the FC components such as adhesion molecules and cytoskeletons to form a mechano-sensing device. However the mechanisms of such channel immobilization on FC remains unclear Tb address this issue, we examined the role of STREX on the channel immobilizatin We expressed GFP-tagged STREX and observed using the single fluorophore video imaging technique. Simultaneous observation with RFP-tagged paxillin showed that STREX molecules were recruited and diffused rapidly on the plasma membrane but significant fraction of them was immobilized on FCs, indicating that STREX itself could associate with certain FC components. However, STREX-deleted SAKcaCs were also immobilized on or near FCs, indicating that SAKcaCs could be associated with Its via STEER and/or other sites of the channel