Abstract
Organic anion transporter 6 [Oat6 (Slc22a20)], a member of the Organic Anion Transporter family uniquely expressed in the testis, mediates the transport of sulfated steroid conjugates. Sequence analysis revealed three putative PKA phosphorylation sites within Oat6. In the current study we analyzed the regulation of PKA on Oat6‐mediated transport using a Chinese Hamster Ovary cell line stably expressing murine Oat6 (CHO‐mOat6). The PKA activator 8‐bromoadenosine (8‐Br‐cAMP) inhibited Oat6‐mediated uptake in a dose and time dependent manner. Preliminary results indicated that pre‐incubation (5 min) of CHO‐mOat6 cells with the PKA inhibitor H‐89 fails to block 8‐Br‐cAMP induced inhibition of Oat6. Using site directed mutagenesis, each PKA site was individually altered and transport activity of the mutants analyzed. Two of the three mutants showed unaltered substrate uptake, whereas the third showed a significant decrease. To confirm expression and plasma membrane targeting of the mutant Oat6 proteins a FLAG tag epitope was fused to Oat6. Introduction of the FLAG tag had no effect on Oat6 transport activity. Immunocytochemistry indicated there was no degradation or mis‐targeting of Oat6 as a result of mutation. These findings suggest that Oat6‐mediated transport of steroid conjugates in the testes may be regulated by PKA through an indirect mechanism.Support: NIDDK RO1‐DK067216, T32 ES012878
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