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MPP+-Induced Changes in Cellular Impedance as a Measure for Organic Cation Transporter (SLC22A1-3) Activity and
Tamara A M Mocking1, Hubert J Sijben1, Yimé W Vermeulen1
1Division of Drug Discovery and Safety, Leiden Academic Centre for Drug Research, Leiden University, Einsteinweg 55, 2333 CC Leiden, The Netherlands.
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.
Insights
A new label-free assay detects organic cation transporter (OCT) activity using cellular impedance and the neurotoxin MPP+. This method identifies OCT inhibitors and potential drug-drug interactions without labeled substrates.
Area of Science:
- Pharmacology
- Biochemistry
- Cell Biology
Background:
- Organic cation transporters (OCTs) 1-3 (SLC22A1-3) are crucial for drug distribution and elimination.
- OCTs' polyspecificity leads to susceptibility to drug-drug interactions (DDIs).
- Current OCT inhibitor screening relies on labeled substrate uptake assays with limitations.
Purpose of the Study:
- To develop a novel, label-free assay for studying OCT-mediated transport and inhibition.
- To establish a method that functions in a physiologically relevant environment without labeled substrates.
- To identify novel OCT inhibitors and potential DDI partners.
Main Methods:
- Development of a label-free impedance-based transport assay.
- Utilized the neurotoxin MPP+ as a substrate for OCTs.
- Quantified OCT-mediated transport and inhibition in OCT1-3 overexpressing cells and endogenous OCT3-expressing HeLa cells.
Main Results:
- MPP+ uptake by OCTs caused concentration-dependent changes in cellular impedance.
- Decynium-22, corticosterone, and Tyrosine Kinase inhibitors demonstrated inhibitory effects.
- The assay successfully quantified OCT-mediated MPP+ transport and inhibition.
Conclusions:
- A novel label-free impedance assay effectively detects OCT-mediated transport and inhibition.
- This method provides a valuable tool for identifying OCT inhibitors and DDI candidates.
- The assay is applicable to MPP+ transporting solute carrier proteins (SLCs) generally.
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