Department of Physiology, Semmelweis University, 1094 Budapest, Hungary.
HUN-REN-SU Molecular Physiology Research Group, Hungarian Research Network and Semmelweis University, 1094 Budapest, Hungary.
Int J Mol Sci. 2024 Aug 15;25(16):8879. doi: 10.3390/ijms25168879.
The TRESK (K2P18.1, KCNK18) background potassium channel is expressed in primary sensory neurons and has been reported to contribute to the regulation of pain sensations. In the present study, we examined the interaction of TRESK with NDFIP1 (Nedd4 family-interacting protein 1) in the oocyte expression system by two-electrode voltage clamp and biochemical methods. We showed that the coexpression of NDFIP1 abolished the TRESK current under the condition where the other K channels were not affected. Mutations in the three PPxY motifs of NDFIP1, which are responsible for the interaction with the Nedd4 ubiquitin ligase, prevented a reduction in the TRESK current. Furthermore, the overexpression of a dominant-negative Nedd4 construct in the oocytes coexpressing TRESK with NDFIP1 partially reversed the down-modulating effect of the adaptor protein on the K current. The biochemical data were also consistent with the functional results. An interaction between epitope-tagged versions of TRESK and NDFIP1 was verified by co-immunoprecipitation experiments. The coexpression of NDFIP1 with TRESK induced the ubiquitination of the channel protein. Altogether, the results suggest that TRESK is directly controlled by and highly sensitive to the activation of the NDFIP1-Nedd4 system. The NDFIP1-mediated reduction in the TRESK component may induce depolarization, increase excitability, and attenuate the calcium dependence of the membrane potential by reducing the calcineurin-activated fraction in the ensemble background K current.
TRESK（K2P18.1，KCNK18）背景钾通道在初级感觉神经元中表达，据报道它参与了痛觉的调节。在本研究中，我们通过双电极电压钳和生化方法在卵母细胞表达系统中检查了 TRESK 与 NDFIP1（Nedd4 家族相互作用蛋白 1）的相互作用。我们表明，在不影响其他 K 通道的情况下，NDFIP1 的共表达消除了 TRESK 电流。负责与 Nedd4 泛素连接酶相互作用的 NDFIP1 的三个 PPxY 基序中的突变阻止了 TRESK 电流的减少。此外，在共表达 TRESK 和 NDFIP1 的卵母细胞中过表达显性负性 Nedd4 构建体部分逆转了衔接蛋白对 K 电流的下调作用。生化数据也与功能结果一致。通过共免疫沉淀实验验证了 TRESK 和 NDFIP1 的表位标记版本之间的相互作用。NDFIP1 与 TRESK 的共表达诱导了通道蛋白的泛素化。总之，这些结果表明 TRESK 直接受 NDFIP1-Nedd4 系统的激活控制且对其高度敏感。NDFIP1 介导的 TRESK 组成部分的减少可能通过降低钙调神经磷酸酶激活的背景 K 电流分数来诱导去极化、增加兴奋性并减弱膜电位对钙的依赖性。
