Identification and Characterization of Cellular Manganese Efflux Mechanisms

Thesis (Ph.D.)--University of Rochester. School of Medicine & Dentistry. Dept. of Environmental Medicine, 2011.Manganese (Mn) is a ubiquitous trace element that is required for life; however, exposures to high levels of Mn can have severe adverse consequences, including the development of a Parkinson’s-like disease known as manganism. The mechanisms by which Mn is transported across cell membranes to reach its target sites are now beginning to be elucidated, although much remains to be learned about these processes, and in particular about cellular export mechanisms. The overall goal of this project was to identify and characterize cellular mechanisms of Mn efflux. Initial studies examined the hypothesis that the iron exporter ferroportin (FPN1) also mediates cellular Mn export, by studying the ability of FPN1 to transport 54Mn in Xenopus laevis oocytes co-expressing the human divalent metal transporter-1 (DMT1/NRAMP2/SLC11A2). The results demonstrated that Mn is indeed a substrate for FPN1, and that FPN1 may be a multi-specific metal efflux carrier that is both pH and membrane potential-sensitive. When compared to oocytes expressing only DMT1, 54Mn accumulation was lower in oocytes also expressing FPN1. In addition, FPN1-expressing oocytes exhibited a four-fold increase in 54Mn export when compared to control oocytes. Export was concentration-dependent and could be partially cis-inhibited by Fe, Co, and Ni. In addition, transport ability was significantly reduced at pH 5.5, but not at pH 8.5, while incubation in high K media lowered Mn efflux, indicating that Mn export on FPN1 is sensitive to pH and membrane potential. To search for additional Mn transporters, the human genome was searched for proteins with predicted amino acid homology to known or suspected Mn transporters from bacteria, yeast and plants. This in silico homology screen identified twenty-two proteins as candidate Mn transporters, and four of these were screened for their ability to transport 54Mn in Xenopus laevis oocytes co-expressing DMT1. Although the injection of the cRNA for the three ATP proteins failed to alter 54Mn accumulation in Xenopus oocytes, oocytes expressing the zinc transporter-2 (ZNT2/SLC30A2) accumulated much less 54Mn, suggesting that ZNT2 may modulate Mn uptake or efflux. ZNT2 expressing oocytes exported twice as much 54Mn when compared to control oocytes, whereas 54Mn uptake was unaffected. Export from the oocytes could be partially cis-inhibited by 100 M Cd, but not by the other metals tested. In addition, incubation in high K media had no effect on 54Mn efflux in ZNT2 expressing oocytes, indicating that this process is not dependent on the membrane potential. Additional studies examined whether the putative yeast vacuolar metal transporter Ypk9p contributes to Mn transport. Under the conditions tested, a role in Mn transport for Ypk9p was not supported, as accumulation of 54Mn was similar in the parental and ypk9- strains. Overall, these findings provide novel insight into mechanisms of Mn transport and a starting point for future research not only into Mn transport, but also to metal transport in general