FLVCR is a heme export protein that is required for proerythroblast survival (
), macrophage heme-iron recycling, and systemic iron homeostasis (ASH abstract, 2007). However, the mechanism and regulation of export through this and related major facilitator superfamily (MFS) members (e.g., Glut4, GlpT) is unknown. To gain insights into the structure and function of FLVCR, we studied the export of zinc mesoporphyrin (ZnMP), a fluorescent heme analog that traffics like heme and blocks heme oxygenase (HO) activity. NRK cells engineered to express huFLVCR were loaded with ZnMP for 30 min, then incubated at 37°C × 90 min with no carrier protein, albumin, or hemopexin (HPXN). In the absence of carrier protein, no export was seen. Export was near complete in the presence of 152 μM (1%) albumin, but less extensive with 15.2 μM albumin and absent with 1.52 μM albumin. Interestingly, HPXN appeared 100 fold more efficacious in inducing ZnMP export, as the residual ZnMP fluorescence in the presence of 0.152 μM HPXN was equivalent to residual intensity in the presence of 15.2 μM albumin. Comparable results were obtained in studies of human blood macrophages. Heme trafficking was next quantitated using 55Fe-heme and a similar study design (ZnMP was added to block HO activity). In the presence of 1.52 μM albumin, 6% of the 55Fe-heme load was exported, while 52% was exported in the presence of 1.52 μM HPXN. When the albumin concentration was increased 100 fold to 152 μM, 67% was exported. As Kds are ∼10 nM and <1 pM, for albumin and HPXN, respectively, our data argue that the rate of heme export depends on the avidity of this binding. Carrier proteins coordinate the heme-iron through two histidines and stabilize this interaction with multiple aromatic amino acids lining the heme-binding pocket. Of note, there are three conserved histidine residues in FLVCR, two of which (aa 145 and 198) are on adjacent extracellular loops, and based upon known MFS family member structures (
), are potentially optimally located to coordinate the heme-iron exiting the channel. Adjacent transmembrane regions contain several aromatic residues capable of lining a heme pocket. These histidine residues are conserved in human, cat, and mouse FLVCR, but are not conserved in closely related paralogs (
), which lack heme export function. We hypothesize they act as a temporary docking site for heme after it exits through the transport channel and prior to being picked up by HPXN in the circulation, and thus as a structural regulator of export efficiency. We anticipate that when heme binds to this site, the channel is held in the open-out position preventing additional heme from being transported. We are currently using site-directed mutagenesis to generate a mutant FLVCR lacking these two histidines and will test its export function. To our knowledge, comparable regulatory mechanisms have not been previously described for other MFS family members or for transport proteins more broadly. Since HPXN is present in a high concentration in human serum (17–25 μM, similar to transferrin, 22–31 μM), it may function physiologically to facilitate the recycling of heme-iron from macrophages after they ingest senescent red cells, in addition to its role as a scavenger protein for protection from internal bleeding or extravascular hemolysis. By extension, modulation of the serum HPXN concentration might ameliorate the anemia of chronic inflammation.
Disclosure: No relevant conflicts of interest to declare.
2007, The American Society of Hematology