Organic Anion Transporter OAT1 Undergoes Constitutive and Protein Kinase C-regulated Trafficking through a Dynamin- and Clathrin-dependent Pathway

Abstract

Organic anion transporter 1 (OAT1) mediates the body disposition of a diverse array of environmental toxins and clinically important drugs. Therefore, understanding the regulation of this transporter has profound clinical significance. We previously demonstrate that OAT1 activity was down-regulated by activation of protein kinase C (PKC), kinetically revealed as a decrease in the maximum transport velocity V max without significant change in the substrate affinity K m of the transporter. In the current study, we showed that OAT1 constitutively internalized from and recycled back to the plasma membrane, and PKC activation accelerated OAT1 internalization without affecting OAT1 recycling. We further showed that treatment of OAT1-expressing cells with concanavalin A, depletion of K ؉ from the cells, or transfection of dominant negative mutants of dynamin-2 or Eps15 into the cells, all of which block the clathrindependent endocytotic pathway, significantly blocked constitutive and PKC-regulated OAT1 internalization. We finally showed that OAT1 colocalized with transferrin, a marker for clathrin-dependent endocytosis, at the cell surface and in the EEA1-positive early endosomes. Together, our findings demonstrated for the first time that (i) OAT1 constitutively traffics between plasma membrane and recycling endosomes, (ii) PKC activation down-regulates OAT1 activity by altering already existent OAT1 trafficking, and (iii) OAT1 internalization occurs partly through a dynamin-and clathrin-dependent pathway.

Novel Mechanism of Impaired Function of Organic Anion-Transporting Polypeptide 1B3 in Human Hepatocytes: Post-Translational Regulation of OATP1B3 by Protein Kinase C Activation

“…PKC activation decreased surface levels of OAT1, OAT4, OATP2B1, and OATP1A2 without affecting total protein levels of these transport proteins after short-term treatment with a PKC activator for up to 1 hour (Zhou et al, 2007(Zhou et al, , 2011Zhang et al, 2008Zhang et al, , 2010Köck et al, 2010). Rapid downregulation of transport function by PKC activation is associated with increased phosphorylation of OATP2B1 (Köck et al, 2010), the brain glutamate/aspartate transporter GLAST-1 (Conradt and Stoffel, 1997), as well as dopamine (Huff et al, 1997) and serotonin transporters (Jayanthi et al, 2005).…”

“…The function of several human transport proteins is regulated by PKC activation. For example, PKC activation downregulates transport function of organic anion transporter (OAT) 1 (Zhang et al, 2008), OAT3 (Duan et al, 2010), and OAT4 (Zhou et al, 2007;Zhang et al, 2010); OATP2B1 (Köck et al, 2010) and OATP1A2 (Zhou et al, 2011); efflux transport protein P-glycoprotein (P-gp) (Miller et al, 1998); and multidrug resistance protein (MRP)2 (Kubitz et al, 2001). OATP1B3 has putative PKC phosphorylation sites as predicted by Scansite 3 (Obenauer et al, 2003).…”

Organic anion transporters: discovery, pharmacology, regulation and roles in pathophysiology

“…Moreover, PKCa was mobilized to the plasma membrane after angiotensin II treatment, and inhibition of PKCa prevented internalization of hOAT1 [103]. In a complementary study, it was demonstrated that hOAT1 is constitutively internalized and recycled back into the plasma membrane [104]. Activation of PKC increased the internalization of hOAT1 without a concomitant increase in recycling, the net effect of which was a reduction in hOAT1 expression in the plasma membrane in response to PKC activation.…”

“…Activation of PKC increased the internalization of hOAT1 without a concomitant increase in recycling, the net effect of which was a reduction in hOAT1 expression in the plasma membrane in response to PKC activation. Interestingly, inhibition of clathrin-dependent endocytosis reduced hOAT1 internalization, clearly indicating a role for clathrin-coated pits in this process [104]. The studies discussed above demonstrated a consistent suppressive role for PKC, including PKCa, in the regulation of Oat1, but a recent investigation challenged this generalization of PKC's role.…”

“…However, total cortical rOat1 protein was increased, while rOat3 protein was reduced in all cellular fractions. These latter findings suggest that rOat1 and rOat3 may be differentially regulated, and rOat1 may have been internalized in response to ureteral obstruction; a hypothesis which is complemented by more recent studies confirming the ability of OAT1 to cycle from the plasma membrane to the cytosol [103,104]. No other studies have assessed such post-renal obstruction with regard to OAT expression; however, investigations have evaluated the effects of ischemia-reperfusion injury on renal OAT expression/function.…”