Mechanistic Insights into XPR1 Phosphate Export and Brain Calcification - Project Summary Brain calcifications, abnormal calcium phosphate deposits that accumulate in blood vessels of the brain, are prevalent in aging populations and associated with a wide range of neurological symptoms, including migraine, parkinsonism, psychosis, and dementia. While pharmacological treatments can alleviate symptoms, no therapies currently exist to prevent the onset or progression of these calcifications. Primary familial brain calcification (PFBC), also known as Fahr’s syndrome, is an inherited neurological disorder characterized by these pathological deposits. Loss-of-function mutations in XPR1, an inorganic phosphate (Pi) exporter, cause PFBC, establishing a direct link between Pi homeostasis and brain disease. XPR1 functions as a master regulator of cellular Pi balance through its coupling of Pi export to intracellular Pi levels via InsP8, a signaling molecule that accumulates under high Pi conditions and binds the SPX domain of XPR1 to enable its activity. Despite XPR1’s central role in Pi regulation, our understanding of its complex physiological roles and the molecular basis underlying its substrate specificity, gating mechanisms, and precise regulation remains limited, representing a critical knowledge gap in Pi biology and PFBC pathogenesis. Emerging evidence indicates that XPR1 mediates substrate translocation through a unique channel-like pathway, with InsP8 governing its activity through control of an intracellular gate. In addition, a second extracellular gate has been identified, and modulation of XPR1 by protein and lipid partners is essential for its proper function. Together, these insights establish a strong foundation for detailed mechanistic characterization of XPR1 transport function. The overarching goal of this proposal is to test whether XPR1 transports additional substrates beyond Pi and to elucidate the molecular and regulatory mechanisms that control its activity. Using an integrated strategy that combines structural, biochemical, and biophysical approaches, we will first define XPR1’s substrate specificity and selectivity. We will then characterize the mechanisms controlling the intra- and extracellular gates, assessing their individual roles and whether they function in a coordinated manner. Importantly, we will also investigate how critical regulatory factors, including inositol phosphates, the neuronal scaffold protein KIDINS220, and lipid modulators, influence XPR1-mediated export, providing key insight into its functional regulation in cells. This work will significantly advance our molecular understanding of cellular Pi homeostasis and, specifically, how XPR1 dysfunction drives brain calcification and its associated neuropathology. Ultimately, this knowledge will support the rational design of targeted therapeutics aimed at preventing or slowing the formation and progression of brain calcifications in PFBC and related disorders, addressing a major unmet clinical need.
