Functional interactions among retinoschisin and its binding partners

描述（由申请人提供）：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，全球患病率为1：5000至1：20，000。XLRS的表型是由RS 1突变引起的，RS 1是编码视网膜裂素的基因，视网膜裂素是一种参与视网膜细胞组织和粘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，因此早期诊断和潜在的临床干预是非常具有挑战性的。已知视网膜劈裂素与质膜中的各种分子相互作用，包括L型电压门控钙通道<$1D亚基（L-VGCC <$1D）。然而，视网膜裂素如何与其结合伙伴相互作用及其在视网膜中的功能作用尚不完全清楚。了解与retinoschisin相互作用的组分的分子性质将使我们能够更充分地了解retinoschisin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。以前，我们证明了L-VGCC <$1D是鸡视网膜中视网膜裂素的结合伴侣. L-VGCC对于光感受器和其他视网膜神经元中的神经递质释放和细胞内Ca 2+稳态是必不可少的。视网膜裂素结合鸡L-VGCC <$1D的500个氨基酸的N-末端区域，其与人L-VGCC <$1D和L-VGCC <$1F亚基高度保守。在人类中，L-VGCC <$1F突变导致不完全先天性静止性夜盲症（CSNB 2）。我们的发现可以解释为什么XLRS和CSNB 2患者都有类似的锥体反应丧失。我们进一步确定了一个新的结合伙伴的维甲酸，质膜Ca 2 +-ATP酶（PMCA 1），钙泵，是重要的分泌过量的细胞内Ca 2+从光感受器。我们的中心假设是，维甲酸是必要的质膜保留的PMCA 1，有一个功能之间的相互作用，维甲酸，L-VGCC，和PMCA 1调节细胞内Ca 2+稳态的光感受器。我们的研究目标是了解retinoschisin如何与L-VGCCs和PMCA 1相互作用，以及这一动态三人组如何通过以下特定目标调节细胞内Ca 2+稳态：目的1。目的2.确定维甲酸（RS 1）和PMCA 1之间的功能相互作用。确定负责RS 1和PMCA 1/ L-VGCC <$1D之间物理相互作用的分子序列;目的3。确定RS 1、L-VGCC 1D和PMCA 1在细胞内Ca 2+稳态中的功能相互作用。我们希望证明：1。RS 1的功能丧失突变将降低PMCA 1膜保留; 2. RS 1，L-VGCC 1D和PMCA 1的特定分子序列负责其物理相互作用; 3. RS 1、L-VGCC 1D和PMCA 1之间的相互作用有助于细胞内Ca 2+稳态。影响：这项研究计划将揭示retinoschisin如何与其结合伙伴，特别是L-VGCCs和PMCA 1相互作用，以及retinoschisin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.