# Potassium transport by the KdpFABC complex - Equipment Supp

> **NIH NIH R01** · NEW YORK UNIVERSITY SCHOOL OF MEDICINE · 2020 · $61,643

## Abstract

This application is for an administrative supplement to parent grant R01 GM108043 to request an instrument
for conducting transport assays in an efficient and automated manner. The SURFE2R N1 from Nanion
Technologies is the first commercial instrument to implement a Solid Supported Membrane (SSM) with
capacitive coupling for measurement of transport from reconstituted proteoliposomes. In this case,
proteoliposomes will contain KdpFABC, and transport assays with the SURFE2R N1 will complement other in
vitro functional assays to characterize site-specific mutants in order to address mechanistic questions posed in
the parent grant application: i.e., the path that K+ ions take as they traverse the molecule, elements of the
protein complex that are responsible for energetic coupling, whether additional ions (e.g., H+) are co-
transported, and if so with what stoichiometry. The SURFE2R N1 uses SSM technology to overcome the low
flux rate innate to transporters and pumps by adsorbing large numbers of molecules to the relatively large
(3mm) sensor and using capacitive coupling to measure their combined ion flow via high-gain/low-noise
amplifiers routinely employed for patch-clamp technologies. Very low amounts of protein are required (0.1-1g)
and the automation of microfluidics provides not only for high time resolution, but also for programmed series
of experiments to be run without user intervention. Indeed, once loaded with sample, a given sensor can be
used for up to 100 individual experiments. Our current protocol using a stirred cuvette and a conventional
fluorimeter requires 25g of protein for each individual experiment with manual substrate additions and sample
exchange. Thus, the SURFE2R N1 will provide a dramatic gain in throughput. In addition, data analysis
software is included with the SURFE2R N1 which is far more sophisticated than the home-made analysis
software currently applied to data from the fluorimeter. The availability of real-time data from the SURFE2R N1
with high time resolution allows fast binding kinetics to be resolved and routine determination of rate constants,
EC50 and IC50. The performance of this instrument, which was not available at the time of the original
application, has been validated on over 100 diverse targets, most of which are either pumps or secondary
transporters. In preliminary trials on KdpFABC, our samples produced very robust signals that readily led to a
precise ATP titration curve. Based on the suitability of our application, Nanion approved our application for a
six-month loan of a SURFE2R N1 and a brand new instrument was recently shipped to our laboratory, where it
is awaiting installation. This instrument would be available for immediate purchase should this grant application
be approved. The SURFE2R N1 will also be useful for studies of the secondary transporter YiiP under another
R01 grant from Dr. Stokes (GM125081) and a second cross-referenced application for this administrative
supplement prog...

## Key facts

- **NIH application ID:** 10133827
- **Project number:** 3R01GM108043-06S1
- **Recipient organization:** NEW YORK UNIVERSITY SCHOOL OF MEDICINE
- **Principal Investigator:** David L. Stokes
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2020
- **Award amount:** $61,643
- **Award type:** 3
- **Project period:** 2014-09-01 → 2022-07-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10133827

## Citation

> US National Institutes of Health, RePORTER application 10133827, Potassium transport by the KdpFABC complex - Equipment Supp (3R01GM108043-06S1). Retrieved via AI Analytics 2026-05-24 from https://api.ai-analytics.org/grant/nih/10133827. Licensed CC0.

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