Department of Physiology, Semmelweis University, Budapest, Hungary.
PLoS One. 2018 May 15;13(5):e0197622. doi: 10.1371/journal.pone.0197622. eCollection 2018.
Two-pore domain K+ channels (K2P) are responsible for background K+ currents and regulate the resting membrane potential and cellular excitability. Their activity is controlled by a large variety of physicochemical factors and intracellular signaling pathways. The majority of these effects converge on the intracellular C-terminus of the channels, resulting in the modification of the gating at the selectivity filter. Another gating mechanism, the activation gate at the helix bundle crossing is also well documented in other K+ channel families, however, it remains uncertain whether this type of gating is functional in K2P channels. The regulation of TWIK-related spinal cord K+ channel (TRESK) is different from the other K2P channels. Regulatory factors acting via the C-terminus are not known, instead channel activity is modified by the phosphorylation/dephosphorylation of the unusually long intracellular loop between the 2nd and 3rd transmembrane segments. These unique structural elements of the regulation lead us to examine channel gating at the bundle crossing region. Ba2+ was applied to the intracellular side of excised membrane patches and the characteristics of the channel block were determined. We compared the kinetics of the development of Ba2+ block when the channels were phosphorylated (inhibited) or dephosphorylated (activated) and also in different mutants mimicking the two functional states. Neither the phosphorylation/dephosphorylation nor the point mutations influenced the development of Ba2+ block, suggesting that the conformational changes of the bundle crossing region do not contribute to the phosphorylation-dependent gating of TRESK.
双孔域钾离子通道（K2P）负责背景钾离子电流，并调节静息膜电位和细胞兴奋性。它们的活动受多种物理化学因素和细胞内信号通路的控制。这些效应中的大多数都集中在通道的细胞内 C 末端，导致选择性过滤器的门控发生变化。另一种门控机制，即螺旋束交叉处的激活门，在其他钾离子通道家族中也有很好的记录，但在 K2P 通道中是否存在这种类型的门控仍不确定。TWIK 相关脊髓钾离子通道（TRESK）的调节不同于其他 K2P 通道。通过 C 末端起作用的调节因子尚不清楚，相反，通道活性通过第 2 和第 3 跨膜片段之间的细胞内环的磷酸化/去磷酸化来修饰。这种调节的独特结构元素促使我们检查束交叉区域的通道门控。将 Ba2+应用于膜片钳的细胞内侧，并确定通道阻断的特征。我们比较了通道磷酸化（抑制）或去磷酸化（激活）时 Ba2+阻断的发展动力学，以及模拟两种功能状态的不同突变体。磷酸化/去磷酸化或点突变都没有影响 Ba2+阻断的发展，这表明束交叉区域的构象变化不参与 TRESK 的磷酸化依赖性门控。
