Kim Dong-Il, Park Yongsoo, Jang Deok-Jin, Suh Byung-Chang
Department of Brain Science, Daegu Gyeongbuk Institute of Science and Technology (DGIST), Daegu 711-873, Korea.
Department of Neurobiology, Max Planck Institute for Biophysical Chemistry, 37077 Göttingen, Germany.
J Gen Physiol. 2015 Jun;145(6):529-41. doi: 10.1085/jgp.201411349. Epub 2015 May 11.
High voltage-activated Ca2+ (Ca(V)) channels are protein complexes containing pore-forming α1 and auxiliary β and α2δ subunits. The subcellular localization and membrane interactions of the β subunits play a crucial role in regulating Ca(V) channel inactivation and its lipid sensitivity. Here, we investigated the effects of membrane phosphoinositide (PI) turnover on Ca(V)2.2 channel function. The β2 isoform β2e associates with the membrane through electrostatic and hydrophobic interactions. Using chimeric β subunits and liposome-binding assays, we determined that interaction between the N-terminal 23 amino acids of β2e and anionic phospholipids was sufficient for β2e membrane targeting. Binding of the β2e subunit N terminus to liposomes was significantly increased by inclusion of 1% phosphatidylinositol 4,5-bisphosphate (PIP2) in the liposomes, suggesting that, in addition to phosphatidylserine, PIs are responsible for β2e targeting to the plasma membrane. Membrane binding of the β2e subunit slowed Ca(V)2.2 current inactivation. When membrane phosphatidylinositol 4-phosphate and PIP2 were depleted by rapamycin-induced translocation of pseudojanin to the membrane, however, channel opening was decreased and fast inactivation of Ca(V)2.2(β2e) currents was enhanced. Activation of the M1 muscarinic receptor elicited transient and reversible translocation of β2e subunits from membrane to cytosol, but not that of β2a or β3, resulting in fast inactivation of Ca(V)2.2 channels with β2e. These results suggest that membrane targeting of the β2e subunit, which is mediated by nonspecific electrostatic insertion, is dynamically regulated by receptor stimulation, and that the reversible association of β2e with membrane PIs results in functional changes in Ca(V) channel gating. The phospholipid-protein interaction observed here provides structural insight into mechanisms of membrane-protein association and the role of phospholipids in ion channel regulation.
高电压激活的Ca2+（Ca(V)）通道是由形成孔道的α1以及辅助性β和α2δ亚基组成的蛋白质复合物。β亚基的亚细胞定位和膜相互作用在调节Ca(V)通道失活及其脂质敏感性方面起着关键作用。在此，我们研究了膜磷酸肌醇（PI）周转对Ca(V)2.2通道功能的影响。β2亚型β2e通过静电和疏水相互作用与膜结合。利用嵌合β亚基和脂质体结合试验，我们确定β2e的N端23个氨基酸与阴离子磷脂之间的相互作用足以实现β2e的膜靶向。通过在脂质体中加入1%的磷脂酰肌醇4,5-二磷酸（PIP2），β2e亚基N端与脂质体的结合显著增加，这表明除了磷脂酰丝氨酸外，PI也负责β2e靶向质膜。β2e亚基的膜结合减缓了Ca(V)2.2电流的失活。然而，当雷帕霉素诱导假贾宁转位到膜上导致膜磷脂酰肌醇4-磷酸和PIP2耗尽时，通道开放减少，Ca(V)2.2(β2e)电流的快速失活增强。M1毒蕈碱受体的激活引发β2e亚基从膜到胞质溶胶的瞬时和可逆转位，但β2a或β3亚基则不会，导致具有β2e的Ca(V)2.2通道快速失活。这些结果表明，由非特异性静电插入介导的β2e亚基的膜靶向受受体刺激动态调节，并且β2e与膜PI的可逆结合导致Ca(V)通道门控的功能变化。此处观察到的磷脂-蛋白质相互作用为膜-蛋白质结合机制以及磷脂在离子通道调节中的作用提供了结构上的见解。
