Institut für Experimentelle und Klinische Pharmakologie und Toxikologie, Fakultät für Medizin, Albert-Ludwigs-Universität Freiburg, Albertstr. 25, 79104, Freiburg, Germany.
Center of Bioscience, Institute for Molecular Physiology and Genetics, 84005, Bratislava, Slovakia.
Pflugers Arch. 2019 Jun;471(6):861-874. doi: 10.1007/s00424-018-02248-x. Epub 2019 Jan 5.
Voltage-gated Ca channels are embedded in a network of protein interactions that are fundamental for channel function and modulation. Different strategies such as high-resolution quantitative MS analyses and yeast-two hybrid screens have been used to uncover these Ca channel nanodomains. We applied the yeast split-ubiquitin system with its specific advantages to search for interaction partners of the Ca2.2 Ca channel and identified four proteins: reticulon 1 (RTN1), member 1 of solute carrier family 38 (SLC38), prostaglandin D2 synthase (PTGDS) and transmembrane protein 223 (TMEM223). Interactions were verified using the yeast split-ubiquitin system and narrowed down to Ca2.2 domain IV. Colocalization studies using fluorescent constructs demonstrated defined regions of subcellular localization. Detailed electrophysiological studies revealed that coexpression of RTN1 modulated Ca2.2 channels only to a minor extent. SLC38 accelerated the cumulative current inactivation during a high-frequency train of brief depolarizing pulses. As neurons expressing Ca2.2 channels were exposed to high-frequency bursts under physiological conditions, observed regulation may have a negative modulatory effect on transmitter release. Coexpression of PTGDS significantly lowered the average current density and slowed the kinetics of cumulative current inactivation. Since the latter effect was not significant, it may only partly compensate the first one under physiological conditions. Expression of TMEM223 lowered the average current density, accelerated the kinetics of cumulative current inactivation and slowed the kinetics of recovery from inactivation. Therefore, TMEM223 and, to a lesser extent, PTGDS, may negatively modulate Ca entry required for transmitter release and/or for dendritic plasticity under physiological conditions.
电压门控钙通道嵌入在蛋白质相互作用网络中，这对于通道功能和调节至关重要。已经使用了不同的策略，如高分辨率定量 MS 分析和酵母双杂交筛选，以揭示这些钙通道纳米域。我们应用了具有其特定优势的酵母分裂泛素系统来搜索 Ca2.2 钙通道的相互作用伙伴，并鉴定出了四种蛋白质：内质网 1（RTN1）、溶质载体家族 38（SLC38）成员 1、前列腺素 D2 合酶（PTGDS）和跨膜蛋白 223（TMEM223）。使用酵母分裂泛素系统验证了相互作用，并将其缩小到 Ca2.2 结构域 IV。使用荧光构建体进行的共定位研究表明存在明确的亚细胞定位区域。详细的电生理研究表明，RTN1 的共表达仅对 Ca2.2 通道产生微小的调制作用。SLC38 加速了在短暂去极化脉冲高频串中的累积电流失活。由于表达 Ca2.2 通道的神经元在生理条件下暴露于高频爆发中，观察到的调节可能对递质释放产生负调节作用。PTGDS 的共表达显著降低了平均电流密度并减缓了累积电流失活的动力学。由于后一种效应不显著，因此在生理条件下，它可能仅部分补偿前一种效应。TMEM223 的表达降低了平均电流密度，加速了累积电流失活的动力学，并减缓了失活后的恢复动力学。因此，TMEM223 和在较小程度上 PTGDS，可能在生理条件下负调节递质释放和/或树突可塑性所需的钙内流。
