MPP+-Induced Changes in Cellular Impedance as a Measure for Organic Cation Transporter (SLC22A1-3) Activity and Inhibition.

Tamara Mocking,Yimé Vermeulen,Hubert Sijben,Laura Heitman,Adriaan Ijzerman

doi:10.3390/ijms23031203

Abstract

The organic cation transporters OCT1-3 (SLC22A1-3) facilitate the transport of cationic endo- and xenobiotics and are important mediators of drug distribution and elimination. Their polyspecific nature makes OCTs highly susceptible to drug–drug interactions (DDIs). Currently, screening of OCT inhibitors depends on uptake assays that require labeled substrates to detect transport activity. However, these uptake assays have several limitations. Hence, there is a need to develop novel assays to study OCT activity in a physiological relevant environment without the need to label the substrate. Here, a label-free impedance-based transport assay is established that detects OCT-mediated transport activity and inhibition utilizing the neurotoxin MPP+. Uptake of MPP+ by OCTs induced concentration-dependent changes in cellular impedance that were inhibited by decynium-22, corticosterone, and Tyrosine Kinase inhibitors. OCT-mediated MPP+ transport activity and inhibition were quantified on both OCT1-3 overexpressing cells and HeLa cells endogenously expressing OCT3. Moreover, the method presented here is a valuable tool to identify novel inhibitors and potential DDI partners for MPP+ transporting solute carrier proteins (SLCs) in general.

Highlights

To maintain cellular homeostasis, cells depend on transporters for the uptake and export of various solutes such as neurotransmitters, hormones, nutrients, and metabolites.Bidirectional transport of cationic endo- and xenobiotics across the cell membrane is facilitated by the organic cation transporters (OCTs) that belong to the SLC22 family
OCT1 and OCT2 are predominantly expressed in the liver and kidney, respectively, while OCT3 is expressed throughout the body [3]
To investigate if OCT-mediated uptake of the neurotoxic substrate MPP+ led to distinct changes in cellular impedance, a HEK293-JumpIn cell-line was used with doxycycline-inducible OCT3 expression (HEK293-JI-OCT3)

Summary

Introduction

Cells depend on transporters for the uptake and export of various solutes such as neurotransmitters, hormones, nutrients, and metabolites.Bidirectional transport of cationic endo- and xenobiotics across the cell membrane is facilitated by the organic cation transporters (OCTs) that belong to the SLC22 family. Cells depend on transporters for the uptake and export of various solutes such as neurotransmitters, hormones, nutrients, and metabolites. OCTs (OCT1-3, SLC22A1-3) are low-affinity, high-capacity transporters of neurotransmitters and other cationic molecules such as metabolites and drugs [1,2]. The ability to transport and interact with structurally diverse molecules designates OCTs as regulators of drug disposition and excretion [4]. Their lack of specificity and the myriad of cationic drugs on the market, e.g., beta-blockers, antihistamines, and antidepressants [5,6], make OCTs highly susceptible for drug-drug interactions (DDIs). DDIs could alter a drug’s pharmacokinetics and pharmacodynamics, and thereby hamper drug effectiveness or lead to adverse events [7]

Methods

Results

Conclusion