Structure-Function of the Human Copper Transporter hCTR1

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DESCRIPTION (provided by applicant): Copper is essential for life and is utilized in critical biological reactions including respiration, clearance of free radicals and iron uptake. Abnormal action of copper ions has been implicated in the neurodegenerative Menkes and Alzheimer's diseases. Despite the importance of copper in biology, surprisingly little is known about the transporters involved in the initial uptake of copper into cells. The recently discovered copper transporter CTR1 is the only known high-affinity copper uptake transporter in humans. CTR1 is ubiquitously expressed in humans and can functionally substitute for the yeast copper transporter Ctrlp. CTR1 and all its homologs are unique among transition metal transporters in having the least number of predicted transmembrane domains per monomer strongly suggesting that oligomerization is required for function. CTR1 is also unique in having an extracellular N-terminal domain that is likely to capture copper ions prior to transport. Human CTR1 (hCTR1) was shown to be a high affinity copper uptake transporter in yeast genetics experiments but has not been characterized at the molecular level. I propose experiments that will demonstrate actual copper transport through hCTR1 in liposomes and Xenopus oocytes to determine the affinity and selectivity of hCTR1 for copper transport. The role of the N-terminal domain of hCTR1 will be determined using copper binding assays and chimeric constructs of hCTR1 with a related low-affinity copper transporter. Purification and reconstitution of hCTR1 in liposomes will be employed to determined the specificity and kinetics of transport. Two-dimensional crystallization of hCTR1 will be initiated to allow structural solution of hCTR1 by cryoelectron microscopy for visualization of pore forming regions, oligomeric state and symmetry in the lipid bilayer. These experiments will initiate the molecular characterization of the unique human CTR1 high-affinity copper transporter and determine the mechanisms by which this protein transports copper through the lipid bilayer.