The direct cause of free sialic acid storage disorder is the genetic mutation in the lysosomal sialic acid transporter Sialin. Although the disease has been discovered for over 20 years, no cure has been developed. The long-term goal is to help create valuable therapeutics for patients with pathogenic Sialin mutations. The overall objective of this proposal is to establish a basic understanding of the membrane transporter. The rationale is that, with detailed knowledge about the structure-function relationship of Sialin, it will be possible to design small molecules to restore the substrate translocation of dysfunctional Sialin. To accomplish the goal, three specific aims will be pursued: 1) understand the substrate translocation mechanism of Sialin; 2) probe the connection between defective Sialin and cardiovascular diseases; and 3) restore the transporter function of pathogenic Sialin mutants. Specifically, in the first aim, biochemical assays, cryo-electron microscopy, and molecular dynamics simulation will reveal the molecular mechanism. In the second aim, Sialin will be characterized in human cardiac cells and a human induced pluripotent stem cell-based platform. In the third aim, small molecules to rescue dysfunctional Sialin will be identified by high-throughput screening and confirmed by secondary and tertiary assays. The research proposal is innovative because it focuses on finding a new strategy to rescue the normal function of pathogenic Sialin mutants that are directly connected with human diseases. This proposal is significant because it is expected to reveal the basic molecular mechanisms of Sialin and provide scientific justification for further development of drugs against various pathogenic Sialin mutations.