Department of Brain & Cognitive Sciences, Daegu Gyeongbuk Institute of Science and Technology, 42988 Daegu, South Korea.
Department of Physiology and Biophysics, University of Washington, Seattle, WA 98195
Proc Natl Acad Sci U S A. 2018 Oct 16;115(42):E9934-E9943. doi: 10.1073/pnas.1809762115. Epub 2018 Sep 26.
β subunits of high voltage-gated Ca (Ca) channels promote cell-surface expression of pore-forming α1 subunits and regulate channel gating through binding to the α-interaction domain (AID) in the first intracellular loop. We addressed the stability of Ca α1B-β interactions by rapamycin-translocatable Ca β subunits that allow drug-induced sequestration and uncoupling of the β subunit from Ca2.2 channel complexes in intact cells. Without Ca α1B/α2δ1, all modified β subunits, except membrane-tethered β2a and β2e, are in the cytosol and rapidly translocate upon rapamycin addition to anchors on target organelles: plasma membrane, mitochondria, or endoplasmic reticulum. In cells coexpressing Ca α1B/α2δ1 subunits, the translocatable β subunits colocalize at the plasma membrane with α1B and stay there after rapamycin application, indicating that interactions between α1B and bound β subunits are very stable. However, the interaction becomes dynamic when other competing β isoforms are coexpressed. Addition of rapamycin, then, switches channel gating and regulation by phosphatidylinositol 4,5-bisphosphate [PI(4,5)P] lipid. Thus, expression of free β isoforms around the channel reveals a dynamic aspect to the α1B-β interaction. On the other hand, translocatable β subunits with AID-binding site mutations are easily dissociated from Ca α1B on the addition of rapamycin, decreasing current amplitude and PI(4,5)P sensitivity. Furthermore, the mutations slow Ca2.2 current inactivation and shift the voltage dependence of activation to more positive potentials. Mutated translocatable β subunits work similarly in Ca2.3 channels. In sum, the strong interaction of Ca α1B-β subunits can be overcome by other free β isoforms, permitting dynamic changes in channel properties in intact cells.
高电压门控 Ca（Ca）通道的β亚基促进孔形成α1亚基的细胞表面表达，并通过与第一个细胞内环中的α相互作用域（AID）结合来调节通道门控。我们通过雷帕霉素可转运的 Caβ亚基来解决 Caα1B-β相互作用的稳定性问题，该亚基允许在完整细胞中通过药物诱导将β亚基与 Ca2.2 通道复合物隔离和解偶。没有 Caα1B/α2δ1，除了膜锚定的β2a 和β2e 之外，所有修饰的β亚基都在细胞质中，并且在雷帕霉素添加到靶细胞器的锚定点时迅速转运：质膜、线粒体或内质网。在共表达 Caα1B/α2δ1 亚基的细胞中，可转运的β亚基与α1B 在质膜处共定位，并在雷帕霉素应用后仍留在那里，这表明α1B 和结合的β亚基之间的相互作用非常稳定。然而，当共表达其他竞争的β同工型时，相互作用变得动态。因此，雷帕霉素的添加会切换通道门控和磷脂酰肌醇 4,5-二磷酸 [PI(4,5)P] 脂质的调节。因此，通道周围游离β同工型的表达揭示了α1B-β相互作用的动态方面。另一方面，与 AID 结合位点突变的可转运β亚基在添加雷帕霉素时很容易与 Caα1B 解离，从而降低电流幅度和 PI(4,5)P 敏感性。此外，突变会减缓 Ca2.2 电流失活并将激活的电压依赖性转移到更正的电位。突变的可转运β亚基在 Ca2.3 通道中也具有相似的作用。总之，Caα1B-β亚基的强相互作用可以被其他游离β同工型克服，从而允许在完整细胞中动态改变通道特性。
