Navarro-Retamal Carlos, Schott-Verdugo Stephan, Gohlke Holger, Dreyer Ingo
Centro de Bioinformática, Simulación y Modelado (CBSM), Facultad de Ingeniería, Campus Talca, Universidad de Talca, Talca 346000, Chile.
John von Neumann Institute for Computing (NIC), Forschungszentrum Jülich GmbH, 52425 Jülich, Germany.
Int J Mol Sci. 2021 Sep 26;22(19):10345. doi: 10.3390/ijms221910345.
Two Pore Channels (TPCs) are cation-selective voltage- and ligand-gated ion channels in membranes of intracellular organelles of eukaryotic cells. In plants, the TPC1 subtype forms the slowly activating vacuolar (SV) channel, the most dominant ion channel in the vacuolar membrane. Controversial reports about the permeability properties of plant SV channels fueled speculations about the physiological roles of this channel type. TPC1 is thought to have high Ca permeability, a conclusion derived from relative permeability analyses using the Goldman-Hodgkin-Katz (GHK) equation. Here, we investigated in computational analyses the properties of the permeation pathway of TPC1 from . Using the crystal structure of AtTPC1, protein modeling, molecular dynamics (MD) simulations, and free energy calculations, we identified a free energy minimum for Ca, but not for K, at the luminal side next to the selectivity filter. Residues D269 and E637 coordinate in particular Ca as demonstrated in in silico mutagenesis experiments. Such a Ca-specific coordination site in the pore explains contradicting data for the relative Ca/K permeability and strongly suggests that the Ca permeability of SV channels is largely overestimated from relative permeability analyses. This conclusion was further supported by in silico electrophysiological studies showing a remarkable permeation of K but not Ca through the open channel.
双孔通道（TPCs）是真核细胞内细胞器膜中的阳离子选择性电压门控和配体门控离子通道。在植物中，TPC1亚型形成缓慢激活的液泡（SV）通道，这是液泡膜中最主要的离子通道。关于植物SV通道通透性特性的争议性报道引发了对这种通道类型生理作用的猜测。TPC1被认为具有高钙通透性，这一结论来自于使用戈德曼-霍奇金- Katz（GHK）方程进行的相对通透性分析。在此，我们通过计算分析研究了拟南芥TPC1渗透途径的特性。利用AtTPC1的晶体结构、蛋白质建模、分子动力学（MD）模拟和自由能计算，我们在选择性过滤器旁边的腔侧确定了一个钙的自由能最小值，而钾没有。如计算机诱变实验所示，残基D269和E637特别配位钙。孔中这种特定于钙的配位位点解释了相对钙/钾通透性的矛盾数据，并强烈表明从相对通透性分析中对SV通道的钙通透性估计过高。计算机电生理研究进一步支持了这一结论，该研究表明钾而非钙通过开放通道有显著的渗透。
