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A Fluorescence-Based Assay of Membrane Potential for High-Throughput Functional Study of Two Endogenous Ion Channels in Two Epithelial Cell Lines
Published on: June 22, 2022
High-throughput fluorescent-based NKCC functional assay in adherent epithelial cells.
Monica Carmosino1, Federica Rizzo, Silvia Torretta
1Department of Biosciences, Biotechnologies and Biopharmaceutics, University of Bari, Via Amendola 165/A, Bari 70126, Italy. monica.carmosino@uniba.it
BMC Cell Biology
|March 20, 2013
Summary
A new fluorescent assay accurately measures NKCC transporter activity, aiding the development of novel anti-hypertensive drugs targeting blood pressure regulation.
Area of Science:
- Biochemistry
- Physiology
- Pharmacology
Background:
- The kidney-specific NKCC2 cotransporter is vital for Na+ reabsorption in the Thick Ascending Limb (TAL) and body fluid volume regulation.
- NKCC1 is crucial for Cl- entry in endothelial cells, impacting cell volume and vascular tone.
- Both NKCC isoforms are key regulators of blood pressure and potential therapeutic targets for hypertension.
Purpose of the Study:
- To develop and validate a novel, high-throughput fluorescent assay for measuring NKCC cotransporter activity.
- To assess the feasibility of this assay in renal epithelial cells for drug screening.
Main Methods:
- Utilized a Thallium (Tl(+))-based kit to establish a Tl(+) influx fluorescent assay.
- Employed a high-throughput Flex station device for rapid assessment of transporter activity.
- Validated the assay in LLC-PK1 cells expressing a chimeric NKCC2 construct (c-NKCC2).
Main Results:
- The developed assay accurately and rapidly measures NKCC transporter activity in adherent epithelial cells.
- Demonstrated high reproducibility and temporal resolution of NKCC-mediated ion flux.
- The continuous nature of the assay offers improved sensitivity compared to previous endpoint assays.
Conclusions:
- The established fluorescence-based assay provides a sensitive and high-throughput method for screening NKCC transporter activity.
- This method is highly valuable for the development and characterization of new anti-hypertensive drugs.
- Offers an advancement over traditional (86)Rb(+) influx assays for NKCC inhibitor screening.
