Structure and mechanism of the kidney disease-causing protein APOL1

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PROJECT SUMMARY In the United States, people of recent African ancestry are four times more likely to develop chronic kidney disease (CKD) and end stage kidney disease (ESKD) than non-Hispanic whites. While some of this risk can be attributed to differential access to care and social determinants of health, much of this excess risk can be attributed to two “high-risk” variants in the Apolipoprotein L1 (APOL1) gene. Patients with APOL1-associated kidney disease are diagnosed with CKD at younger ages, have more rapid CKD progression, and are less likely to respond to existing therapies. Although the link between the high-risk APOL1 genotype and the increased risk of kidney disease is well-established, basic questions about the structure of the APOL1 protein and the differences in activity between the high- and low-risk variants remain unanswered. Purified bacterially- expressed APOL1 proteins have been observed to have nonselective cation channel activity. However, the oligomerization state of the active APOL1 channel and the structural determinants of its selectivity are unknown. Furthermore, the functional effects of the high-risk variants on APOL1 channel activity are unknown. In preliminary work, I have purified eukaryotically-expressed, recombinant APOL1 proteins and shown that they can form active ion channels in vitro. I hypothesize that APOL1 forms oligomeric nonselective cation channels, and that channels composed of high-risk variant APOL1 proteins permit greater cation currents than channels composed of the low-risk variant protein. To test this hypothesis, I propose two specific aims. In Aim 1, I will solve the structure of the APOL1 protein using electron microscopy. In Aim 2, I will reconstitute purified APOL1 proteins into liposomes and characterize their activity using fluorometric liposome efflux assays. In pursuing this work, I will gain critical skills and expertise necessary to study the structural and biophysical properties of membrane proteins involved in the pathogenesis of kidney diseases. I will develop as a physician- scientist through formal coursework, scientific programming, and direct mentorship from experts in kidney disease, structural biology, and ion channel physiology. The proposed research aims and career development plan will help me establish a career as an independent investigator who studies the fundamental biochemical and biophysical processes that cause kidney disease, while continuing to treat patients with kidney disease as a nephrologist.