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-VGCC1D). 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- VGCC1D 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-VGCC1D, which is highly conserved with human L-VGCC1D and L-VGCC1F subunits. In humans, mutations of L-VGCC1F 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-VGCC1D; 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-VGCC1D, 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.