Small‐conductance Ca²⁺‐activated K⁺ channel subtype2 (SK2) are stable macromolecular complexes that regulate myocardial excitability and Ca²⁺ homeostasis. Junctophilin‐2 (JP2) is a membrane‐binding protein, which provides functional crosstalk by physically linking with the cell‐surface and intracellular ion channels. We previously demonstrated that the MORN domain of JP2 interacts with SK2 channels. However, the roles of the JP2 MORN domain in regulating the precise subcellular localization and molecular modulation of SK2 have not yet been incompletely understood. In the present study, in vitro and in vivo assays were used to confirm the physical interactions between the SK2 channel and JP2 in H9c2 and HEK293 cells, with a concentration on the association between the C‐terminus of SK2 channels and the MORN domain of JP2. Furthermore, the membrane expression of SK2 were found to be significantly impaired by the mutation or knockdown of JP2. Using immunofluorescence staining along with Golgi/early endosome markers, we studied the mechanisms of JP2‐regulated SK2 membrane trafficking, which indicates that the JP2 MORN domain is probably necessary for the retrograde trafficking of SK2 channels. The functional study demonstrates that whole cell SK2 current densities recorded from the HEK293 cells co‐expressing the JP2‐MORN domain with SK2 were significantly augmented, compared with cells expressing SK2 alone. Our findings suggest that the MORN domain of JP2 directly modulates SK2 channel current amplitude and trafficking, through its interaction with an overlapping region of the JP2 MORN domain on the SK2 C‐terminus.

中文翻译：

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Junctophilin2 的 MORN 结构域调节与小电导 Ca2+ 激活钾通道亚型 2 (SK2) 的功能相互作用


小电导 Ca ²⁺激活的 K ⁺通道亚型 2 (SK2) 是稳定的大分子复合物，可调节心肌兴奋性和 Ca ²⁺稳态。 Junctophilin-2 (JP2) 是一种膜结合蛋白，通过与细胞表面和细胞内离子通道的物理连接提供功能串扰。我们之前证明了 JP2 的 MORN 结构域与 SK2 通道相互作用。然而，JP2 MORN 结构域在调节 SK2 精确亚细胞定位和分子调节中的作用尚未完全了解。在本研究中，使用体外和体内测定来确认H9c2和HEK293细胞中SK2通道和JP2之间的物理相互作用，重点是SK2通道的C末端和JP2的MORN结构域之间的关联。 。此外，发现 JP2 的突变或敲低会显着损害 SK2 的膜表达。使用免疫荧光染色和高尔基体/早期内体标记，我们研究了 JP2 调节 SK2 膜运输的机制，这表明 JP2 MORN 结构域可能是 SK2 通道逆行运输所必需的。功能研究表明，与单独表达 SK2 的细胞相比，共表达 JP2-MORN 结构域和 SK2 的 HEK293 细胞记录的全细胞 SK2 电流密度显着增强。我们的研究结果表明，JP2 的 MORN 结构域通过与 SK2 C 末端 JP2 MORN 结构域的重叠区域相互作用，直接调节 SK2 通道电流幅度和运输。