Shi Liheng, Jian Kuihuan, Ko Michael L, Trump Dorothy, Ko Gladys Y-P
Department of Veterinary Integrative Biosciences, College of Veterinary Medicine and Biomedical Sciences, Texas A&M University, College Station, Texas 77843-4458, USA.
J Biol Chem. 2009 Feb 6;284(6):3966-75. doi: 10.1074/jbc.M806333200. Epub 2008 Dec 11.
The L-type voltage-gated calcium channels (L-VGCCs) are activated under high depolarization voltages. They are vital for diverse biological events, including cell excitability, differentiation, and synaptic transmission. In retinal photoreceptors, L-VGCCs are responsible for neurotransmitter release and are under circadian influences. However, the mechanism of L-VGCC regulation in photoreceptors is not fully understood. Here, we show that retinoschisin, a highly conserved extracellular protein, interacts with the L-VGCCalpha1D subunit and regulates its activities in a circadian manner. Mutations in the gene encoding retinoschisin (RS1) cause retinal disorganization that leads to early onset of macular degeneration. Since ion channel activities can be modulated through interactions with extracellular proteins, disruption of these interactions can alter physiology and be the root cause of disease states. Co-immunoprecipitation and mammalian two-hybrid assays showed that retinoschisin and the N-terminal fragment of the L-VGCCalpha1 subunit physically interacted with one another. The expression and secretion of retinoschisin are under circadian regulation with a peak at night and nadir during the day. Inhibition of L-type VGCCs decreased membrane-bound retinoschisin at night. Overexpression of a missense RS1 mutant gene, R141G, into chicken cone photoreceptors caused a decrease of L-type VGCC currents at night. Our findings demonstrate a novel bidirectional relationship between an ion channel and an extracellular protein; L-type VGCCs regulate the circadian rhythm of retinoschisin secretion, whereas secreted retinoschisin feeds back to regulate L-type VGCCs. Therefore, physical interactions between L-VGCCalpha1 subunits and retinoschisin play an important role in the membrane retention of L-VGCCalpha1 subunits and photoreceptor-bipolar synaptic transmission.
L型电压门控钙通道（L-VGCCs）在高去极化电压下被激活。它们对多种生物学事件至关重要，包括细胞兴奋性、分化和突触传递。在视网膜光感受器中，L-VGCCs负责神经递质释放，并受昼夜节律影响。然而，光感受器中L-VGCC调节的机制尚未完全了解。在这里，我们表明视网膜蛋白（retinoschisin），一种高度保守的细胞外蛋白，与L-VGCCα1D亚基相互作用，并以昼夜节律的方式调节其活性。编码视网膜蛋白（RS1）的基因突变会导致视网膜紊乱，进而导致黄斑变性的早期发作。由于离子通道活性可以通过与细胞外蛋白的相互作用来调节，这些相互作用的破坏会改变生理功能，并成为疾病状态的根本原因。免疫共沉淀和哺乳动物双杂交试验表明，视网膜蛋白和L-VGCCα1亚基的N端片段相互之间存在物理相互作用。视网膜蛋白的表达和分泌受昼夜节律调节，夜间达到峰值，白天降至最低点。抑制L型电压门控钙通道会在夜间减少膜结合的视网膜蛋白。将错义RS1突变基因R141G过表达到鸡视锥光感受器中，会导致夜间L型电压门控钙通道电流减少。我们的研究结果证明了离子通道与细胞外蛋白之间一种新的双向关系；L型电压门控钙通道调节视网膜蛋白分泌的昼夜节律，而分泌的视网膜蛋白则反馈调节L型电压门控钙通道。因此，L-VGCCα1亚基与视网膜蛋白之间的物理相互作用在L-VGCCα1亚基的膜保留和光感受器-双极突触传递中起重要作用。
