Functional interactions among retinoschisin and its binding partners

描述(申请人提供)：X连锁青少年视网膜劈裂症(XLRS)是青少年男性黄斑变性的主要原因，全球发病率从1：5000到1：20,000。XLRS的表型是由RS1基因突变引起的，RS1基因编码视黄素，一种参与视网膜细胞组织和黏附的细胞外八聚体蛋白。由于XLRS的进展和严重程度是高度可变的，早期诊断和潜在的临床干预是非常具有挑战性的。维甲酸与细胞膜上的多种分子相互作用，包括L型电压门控钙通道1D亚单位(L-VGCC？1D)。然而，视黄醇如何与其结合伙伴相互作用以及它在视网膜中的功能作用尚不完全清楚。了解与视黄醇相互作用的成分的分子性质将使我们更充分地了解视黄醇在视网膜细胞生理和功能中的作用，这将为早期诊断和潜在的临床干预提供新的信息。此前，我们证明了L-VGCC？1D是鸡视网膜中视黄醇的结合伙伴。L-VGCC对光感受器和其他视网膜神经元的神经递质释放和细胞内钙稳态是必不可少的。维甲酸与鸡L-VGCC？1D的N-末端500个氨基酸结合，与人L-VGCC？1D和L-VGCC？1F亚基高度保守。在人类中，L基因突变导致不完全性先天性静止性夜盲(CSNB2)。我们的发现可能解释了为什么XLRS和CSNB2患者都有相似的锥体反应丧失。我们进一步确定了视黄醇的一个新的结合伙伴，质膜钙-ATPase(PMCA1)，它是一种钙泵，在从光感受器排泄细胞内过量钙的过程中起重要作用。我们的中心假说是视黄醇对PMCA1的质膜保持是必需的，并且视黄醇、L-VGCC和PMCA1之间存在功能上的相互作用来调节光感受器细胞内的钙稳态。我们的研究目标是了解视黄醇如何与L-VGCCs和PMCA1相互作用，以及这个动态三者如何通过以下特定目的调节细胞内钙稳态：目的1.确定视黄醇与PMCA1之间的功能相互作用；目的2.确定RS1与PMCA1/L-VGCC？1D之间的物理相互作用的分子序列；目的3.确定RS1、L-VGCC1D和PMCA1在细胞内钙稳态中的功能相互作用。我们期望证明：1.RS1功能缺失突变将降低PMCA1膜保留率；2.RS1、L-VGCC1D和PMCA1的特定分子序列负责它们之间的物理相互作用；3.RS1、L-VGCC1D和PMCA1之间的相互作用有助于细胞内钙稳态。影响：本研究计划将揭示视黄醇如何与其结合伙伴，特别是L-VGCCs和PMCA1相互作用，以及视黄醇在光感受器生理和功能中的新作用。

DESCRIPTION (provided by applicant): X-linked juvenile retinoschisis (XLRS) is a leading cause of macular degeneration in juvenile males, with a worldwide prevalence from 1:5000 to 1:20,000. The phenotypes of XLRS are caused by mutations of RS1, the gene encoding retinoschisin, an extracellular octameric protein that participates in retinal cell organization an adhesion. Because XLRS progression and severity is highly variable, early diagnosis and potential clinical intervention is very challenging. Retinoschisin is known to interact with variou molecules in the plasma membrane, including the L-type voltage-gated calcium channel ¿1D subunit (L-VGCC¿1D). However, how retinoschisin interacts with its binding partners and its functional roles in the retina are not completely clear. Understanding the molecular nature of the components that interact with retinoschisin will allow us to more fully understand the roles of retinoschisin in retinal cell physiology and function, which will provide new information for making early diagnosis and potential clinical intervention possible. Previously, we demonstrated that L- VGCC¿1D is a binding partner of retinoschisin in the chick retina. The L-VGCCs are essential for neurotransmitter release and intracellular Ca2+ homeostasis in photoreceptors and other retinal neurons. Retinoschisin binds a 500 amino acid N-terminal region of chicken L-VGCC¿1D, which is highly conserved with human L-VGCC¿1D and L-VGCC¿1F subunits. In humans, mutations of L-VGCC¿1F cause incomplete congenital stationary night blindness (CSNB2). Our finding may explain why both XLRS and CSNB2 patients share a similar loss of cone responses. We have further identified a new binding partner of retinoschisin, the plasma membrane Ca2+-ATPase (PMCA1), a Ca2+ pump that is important in excreting excessive intracellular Ca2+ from photoreceptors. Our central hypothesis is that retinoschisin is necessary for plasma membrane retention of PMCA1, and there is a functional interaction among retinoschisin, L-VGCC, and PMCA1 to regulate intracellular Ca2+ homeostasis in photoreceptors. Our research goal is to understand how retinoschisin interacts with L-VGCCs and PMCA1, and how this dynamic trio regulates intracellular Ca2+ homeostasis through the following specific aims: Aim 1. Determine the functional interaction between retinoschisin (RS1) and PMCA1; Aim 2. Determine the molecular sequences responsible for the physical interactions between RS1 and PMCA1 / L-VGCC¿1D; Aim 3. Determine the functional interaction among RS1, L-VGCC1D, and PMCA1 in intracellular Ca2+ homeostasis. We expect to demonstrate: 1. Loss-of- function mutations of RS1 will decrease PMCA1 membrane retention; 2. Specific molecular sequences of RS1, L-VGCC¿1D, and PMCA1 responsible for their physical interactions; 3. Interactions among RS1, L-VGCC1D, and PMCA1 contribute to intracellular Ca2+ homeostasis. Impact: This research program will reveal how retinoschisin interacts with its binding partners, specifically L-VGCCs and PMCA1, and new roles of retinoschisin in photoreceptor physiology and function.