Related Experiment Video
Updated: Jun 16, 2026
Study of the Functions and Activities of Neuronal K-Cl Co-Transporter KCC2 Using Western Blotting
Published on: December 9, 2022
Rapid modulation of the organic anion transporting polypeptide 2B1 (OATP2B1, SLCO2B1) function by protein kinase
Kathleen Köck1, Anna Koenen, Bernd Giese
1Department of Pharmacology, Research Center of Pharmacology and Experimental Therapeutics, Ernst Moritz Arndt University, 17487 Greifswald, Germany.
Abstract:
Members of the organic anion transporting polypeptide (OATP) family are involved in various pharmacological, pathophysiological, and physiological processes, such as hepatic drug uptake, progress of cancer, or transport of hormones. Although variability in expression and function of OATPs has been investigated in detail, data concerning regulation are rather limited. Here, we report a novel mechanism for rapid regulation of OATP2B1 mediated by protein kinase C (PKC) resulting in significant changes of transport activity. PKC activation by the phorbol ester (phorbol 12-myristate 13-acetate, PMA) resulted in increased phosphorylation of OATP2B1 as well as reduced OATP2B1 transport activity with a decrease in V(max) of E(1)S uptake (288 +/- 21 (control) versus 165 +/- 16 pmol/min/mg of protein (PMA)). This effect was sensitive to the PKC inhibitor bisindolylmaleimide I (BIM-I). Confocal microscopy, fluorescence-based internalization assay, and live-cell imaging using green fluorescent protein-tagged OATP2B1 revealed that transport inhibition was due to internalization of the transporter. Furthermore, colocalization with LAMP-2 and chloroquine-sensitive degradation of OATP2B1 suggest that the internalized protein is targeted to a lysosomal degradation pathway. With regard to the underlying mechanism inhibition of caveolin/lipid raft-mediated endocytosis failed to prevent OATP2B1 internalization, whereas inhibition of clathrin-mediated processes blocked OATP2B1 sequestration. However, small interfering RNA-mediated clathrin knock-down affected general trafficking of OATP2B1 and resulted in intracellular accumulation in the absence of PMA. In conclusion, our data demonstrate that OATP2B1 function is regulated by PKC-mediated, clathrin-dependent internalization and followed by lysosomal degradation. Furthermore, internalization could be shown in an ex vivo placenta perfusion. Our findings represent a new, rapid mechanism in regulation of human OATPs.
Insights
Protein kinase C (PKC) rapidly regulates organic anion transporting polypeptide 2B1 (OATP2B1) function. PKC activation triggers OATP2B1 internalization and lysosomal degradation, impacting drug transport.
Area of Science:
- Pharmacology
- Cell Biology
- Biochemistry
Background:
- Organic anion transporting polypeptides (OATPs) are crucial for drug uptake and hormone transport.
- Regulation mechanisms for OATPs, particularly OATP2B1, are not well understood.
Purpose of the Study:
- To investigate a novel mechanism for rapid regulation of OATP2B1.
- To elucidate the role of protein kinase C (PKC) in OATP2B1 activity.
Main Methods:
- PKC activation using phorbol ester (PMA) and inhibition with bisindolylmaleimide I (BIM-I).
- OATP2B1 phosphorylation and transport activity assays (E1S uptake).
- Confocal microscopy, live-cell imaging (GFP-tagged OATP2B1), and internalization assays.
- Colocalization studies (LAMP-2) and degradation assays (chloroquine).
- Inhibition of endocytosis pathways (caveolin/lipid raft and clathrin-mediated).
- siRNA-mediated clathrin knockdown.
- Ex vivo human placenta perfusion model.
Main Results:
- PKC activation significantly reduced OATP2B1 transport activity (Vmax decreased).
- PMA treatment increased OATP2B1 phosphorylation and led to transporter internalization.
- Internalization was mediated by clathrin-dependent endocytosis, not caveolin/lipid rafts.
- Internalized OATP2B1 was degraded via a lysosomal pathway.
- PKC-mediated OATP2B1 internalization was observed in an ex vivo placenta model.
Conclusions:
- OATP2B1 function is rapidly regulated by PKC-mediated, clathrin-dependent internalization.
- Internalized OATP2B1 is targeted for lysosomal degradation, representing a novel regulatory mechanism.
- This rapid regulation pathway has implications for drug disposition and physiological transport processes.
More Related Videos
Related Concept Videos
01:06Carrier-Mediated Transport
Active transport involves two types of membrane-spanning transporters: uptake and efflux. Uptake transporters are expressed in the small...
01:16Pharmacogenetics of Drug Transporters: P-Glycoprotein and Solute Carrier Transporters
01:16Facilitated Diffusion
In this process, substrates such as organic compounds and ions interact with a transporter on one side, triggering conformational changes in proteins that enable...
01:14Active Transport
Primary active transporters, like Na+, K+ and -ATPase, directly utilize ATP to move ions across the membrane. These transporters play significant roles in various physiological processes. For instance, Na+, K+ and -ATPase maintain...
01:31Membrane Transporters
Transporters are mainly composed of alpha-helices, built from bundles of ten or more helices traversing the plasma membrane. The solute-binding sites are located midway, where some of the helices are broken or distorted, making space for the binding site through...
01:44The Significance of Membrane Transport
Transporters facilitate either an active or passive movement of solutes. They can allow a single-molecule transport down its...