Development of a Label-free Assay for Sodium-Dependent Phosphate Transporter NaPi-IIb

Abstract

The most widely used assay format for characterizing plasma membrane transporter activity measures accumulation of radiolabeled substrates in tissues or cells expressing the transporters. This assay format had limitations and disadvantages; therefore, there was an unmet need for development of a homogeneous, nonradioactive assay for membrane transporter proteins. In this report, the authors describe the development of a label-free homogeneous assay for the sodium-dependent phosphate transporter NaPi-IIb using the Epic system. The addition of phosphate stimulated a dynamic mass redistribution (DMR) profile unique to cells expressing NaPi-IIb but not on parental cells. This DMR profile was phosphate specific because sulfate or buffer alone did not elicit the same response. Furthermore, the DMR response observed was phosphate and sodium dependent, with Km values in the micromolar and millimolar range, respectively. A known NaPi-IIb noncompetitive inhibitor was shown to completely inhibit the phosphate-stimulated DMR response, suggesting that this observed DMR response is an NaPi-IIb-mediated cellular event. The results demonstrate that a novel label-free assay was developed for studying transporter-mediated cellular activity, and this DMR assay platform could be applicable to other membrane transporter proteins.

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

“…Moreover, both assay types are generally "end point" assays, such that time-dependency cannot be easily studied in real-time. Recently label-free techniques, as a new technology, were introduced to study transporter activity [16,19,31,32]. We previously introduced the so-called TRACT assay requiring a GPCR partner to detect SLC-transport activity via a mutual substrate/agonist [17][18][19].…”

Label-free detection of transporter activity via GPCR signalling in living cells: A case for SLC29A1, the equilibrative nucleoside transporter 1

“…Of note, as far as membrane transporters are concerned, there is currently a single report in which an optical-based label-free assay (i.e. Epic by Corning) is used to study the electrogenic transporter SLC34A2 2 . However, this method has not been applied to other electrogenic transporters so far, and still leaves a need for the many non-electrogenic transporters.…”

“…However, their in-depth investigation has been limited as not many cellular assays are available to study transporter activity, making drug discovery difficult. The main and most well-known assay format currently used is the uptake assay, which measures the accumulation of a radiolabeled substrate in cells expressing the transporter under investigation 2 . Such assay however, presents limitations as only the end-point of the assay can be measured and/or washing steps are needed, which make this type of assay laborious and prone to artefacts.…”

An Overview of Cell-Based Assay Platforms for the Solute Carrier Family of Transporters

“…Reports on label-free cell-based transporter assays have been limited ( Wong et al, 2012 ). Recently, a label-free impedance-based assay was developed using xCELLigence to assess functional activity of SLC29A1 (equilibrative nucleoside transporter 1 or ENT1) in living cells ( Vlachodimou et al, 2019 ).…”