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.
Cell Calcium. 2019 Jun;80:71-78. doi: 10.1016/j.ceca.2019.04.002. Epub 2019 Apr 8.
Grina/TMBIM3 is a poorly characterized transmembrane protein with a broad expression pattern in mammals and with a very ancient origin within eukaryotes. Although initially characterized as an NMDA-receptor associated subunit, there is increasing evidence that Grina/TMBIM3 is involved in the unfolded protein response and controls apoptosis via regulation of Ca homeostasis. Here, we investigate a putative direct interaction of Grina/TMBIM3 with voltage gated Ca channels, in particular with the Ca2.2 α1-subunit and describe its modulatory effects on the current through Ca2.2 N-type channels. Direct interaction was confirmed by co-immunoprecipitation studies and membrane localization was proven. Co-expression of Grina/TMBIM3 with Ca2.2 channels resulted in a significant decrease of the current amplitude and in a slowing of the kinetics of current activation. This effect was accompanied by a significant shift of the voltage dependencies of activation time constants towards more depolarized voltages. Application of a stimulus protocol including a strong depolarizing pulse relieved inhibition of current amplitude by Grina/TMBIM3. When Grina/TMBIM3 was present, inactivation by an action potential-like train of pulses was diminished. Both observations resemble mechanisms that are well-studied modulatory effects of G-protein βγ subunits on Ca2 channels. The impact of Grina/TMBIM3 and G-protein βγ subunits are rather comparable with respect to suppression of current amplitude and slowing of activation kinetics. Furthermore, both modulators had the same effect on current inactivation when evoked by an action potential-like train of pulses.
Grina/TMBIM3 是一种特征不明显的跨膜蛋白，在哺乳动物中有广泛的表达模式，并且在真核生物中具有非常古老的起源。尽管最初被描述为 NMDA 受体相关亚基，但越来越多的证据表明 Grina/TMBIM3 参与未折叠蛋白反应，并通过调节 Ca 稳态来控制细胞凋亡。在这里，我们研究了 Grina/TMBIM3 与电压门控 Ca 通道（特别是 Ca2.2 α1 亚基）之间可能存在的直接相互作用，并描述了其对 Ca2.2 N 型通道电流的调节作用。通过共免疫沉淀研究证实了直接相互作用，并证明了膜定位。Grina/TMBIM3 与 Ca2.2 通道的共表达导致电流幅度显著减小，并使电流激活动力学减慢。这种效应伴随着激活时间常数的电压依赖性向更去极化电压的显著偏移。应用包括强去极化脉冲的刺激方案缓解了 Grina/TMBIM3 对电流幅度的抑制作用。当 Grina/TMBIM3 存在时，类似动作电位样脉冲串的失活作用减弱。这两种观察结果都类似于 G 蛋白 βγ 亚基对 Ca2 通道的研究充分的调节作用机制。Grina/TMBIM3 和 G 蛋白 βγ 亚基的影响在抑制电流幅度和减慢激活动力学方面相当可比。此外，当通过类似动作电位样脉冲串诱发时，两种调节剂对电流失活具有相同的作用。
