Characterization of the murine organic anion transporter Oat6 (Slc22a20): Insight to its role in the olfactory mucosa and testes.

摘要

The organic anion transporter (OAT) family mediates the transport of negatively charged drugs, toxins, and endogenous compounds. To date, eleven OAT family members have been identified: OATs 1-10 and URAT1. With the exception of Oat6, all of the OAT family members are identified in either the kidney or liver. Oat6 however demonstrates unique expression as it is identified in the olfactory mucosa and testis. Prior to the studies described in this dissertation, knowledge pertaining to Oat6 was very modest and evidence as to Oat6's role in organic anion transport had not been pursued. Furthermore, virtually nothing is known about organic anion transport in the olfactory mucsosa and testis. In this dissertation studies were performed to characterize the function and regulation of Oat6. Using a stably transfected Oat6 cell line, these studies demonstrated for the first time that Oat6 mediates the transport of the sulfated steroid hormone, estrone sulfate, and therefore functions as an organic anion transporter. In addition, Oat6-mediated transport was found to operate under trans-stimulation, a mechanism employed by Oat1 and Oat3. Analysis of Oat6's interaction with various organic anion substrates and inhibitors revealed unique substrate specificity when compared to other OAT family members. Regulation of OAT mediated transport was analyzed by measuring uptake in the presence of various PKC and PKA activators. While these experiments reveal that neither PKC or PKA is able to directly regulate Oat6 transport, mutation to one of Oat6's three putative PKA sites revealed a ∼37% decrease in Oat6 mediated uptake. Further analysis in the mutant cell line revealed an increase in Km and decrease in Vmax indicating a reduction in transporter number at the cell surface, which was supported using immunocytochemistry to analyze mOat6 expression in the mutant cell line. These results suggest that mutation at this particular site may result in disruption of the Oat6 structure thereby preventing it from translocating to the cell surface. Together, these findings increase our knowledge of Oat6 function and regulation in comparison to other OAT family members and begin to shed light on the process of organic anion transport in the olfactory mucosa and testis.