Vitko Iuliia, Shcheglovitov Aleksandr, Baumgart Joel P, Arias-Olguín Imilla I, Murbartián Janet, Arias Juan Manuel, Perez-Reyes Edward
Department of Pharmacology and Neuroscience Graduate Program, University of Virginia, Charlottesville, Virginia, USA.
PLoS One. 2008;3(10):e3560. doi: 10.1371/journal.pone.0003560. Epub 2008 Oct 29.
The Ca(v)beta subunits of high voltage-activated Ca(2+) channels control the trafficking and biophysical properties of the alpha(1) subunit. The Ca(v)beta-alpha(1) interaction site has been mapped by crystallographic studies. Nevertheless, how this interaction leads to channel regulation has not been determined. One hypothesis is that betas regulate channel gating by modulating movements of IS6. A key requirement for this direct-coupling model is that the linker connecting IS6 to the alpha-interaction domain (AID) be a rigid structure.
METHODOLOGY/PRINCIPAL FINDINGS: The present study tests this hypothesis by altering the flexibility and orientation of this region in alpha(1)2.2, then testing for Ca(v)beta regulation using whole cell patch clamp electrophysiology. Flexibility was induced by replacement of the middle six amino acids of the IS6-AID linker with glycine (PG6). This mutation abolished beta2a and beta3 subunits ability to shift the voltage dependence of activation and inactivation, and the ability of beta2a to produce non-inactivating currents. Orientation of Ca(v)beta with respect to alpha(1)2.2 was altered by deletion of 1, 2, or 3 amino acids from the IS6-AID linker (Bdel1, Bdel2, Bdel3, respectively). Again, the ability of Ca(v)beta subunits to regulate these biophysical properties were totally abolished in the Bdel1 and Bdel3 mutants. Functional regulation by Ca(v)beta subunits was rescued in the Bdel2 mutant, indicating that this part of the linker forms beta-sheet. The orientation of beta with respect to alpha was confirmed by the bimolecular fluorescence complementation assay.
CONCLUSIONS/SIGNIFICANCE: These results show that the orientation of the Ca(v)beta subunit relative to the alpha(1)2.2 subunit is critical, and suggests additional points of contact between these subunits are required for Ca(v)beta to regulate channel activity.
高电压激活的Ca(2+)通道的Ca(v)β亚基控制α(1)亚基的转运和生物物理特性。Ca(v)β-α(1)相互作用位点已通过晶体学研究确定。然而，这种相互作用如何导致通道调节尚未明确。一种假说是β亚基通过调节IS6的运动来调节通道门控。这种直接偶联模型的一个关键要求是连接IS6与α相互作用结构域（AID）的连接子为刚性结构。
方法/主要发现：本研究通过改变α(1)2.2中该区域的灵活性和方向来验证这一假说，然后使用全细胞膜片钳电生理学检测Ca(v)β调节情况。通过用甘氨酸取代IS6-AID连接子中间的六个氨基酸（PG6）来诱导灵活性。该突变消除了β2a和β3亚基改变激活和失活电压依赖性的能力，以及β2a产生非失活电流的能力。通过从IS6-AID连接子中缺失1、2或3个氨基酸（分别为Bdel1、Bdel2、Bdel3）来改变Ca(v)β相对于α(1)2.2的方向。同样，Ca(v)β亚基调节这些生物物理特性的能力在Bdel1和Bdel3突变体中完全丧失。Ca(v)β亚基的功能调节在Bdel2突变体中得以恢复，表明连接子的这部分形成了β折叠。通过双分子荧光互补分析证实了β相对于α的方向。
结论/意义：这些结果表明Ca(v)β亚基相对于α(1)2.2亚基的方向至关重要，并提示这些亚基之间需要额外的接触点才能使Ca(v)β调节通道活性。
