Tuning the sensitivity of genetically encoded fluorescent potassium indicators through structure-guided and genome mining strategies

Torres Cabán, C. C., Yang, M., Lai, C., Yang, L., Subach, F. V., Smith, B. O. , Piatkevich, K. D. and Boyden, E. S. (2022) Tuning the sensitivity of genetically encoded fluorescent potassium indicators through structure-guided and genome mining strategies. ACS Sensors, 7(5), pp. 1336-1346. (doi: 10.1021/acssensors.1c02201) (PMID:35427452) (PMCID:PMC9150168)

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Abstract

Genetically encoded potassium indicators lack optimal binding affinity for monitoring intracellular dynamics in mammalian cells. Through structure-guided design and genome mining of potassium binding proteins, we developed green fluorescent potassium indicators with a broad range of binding affinities. KRaION1 (K+ ratiometric indicator for optical imaging based on mNeonGreen 1), based on the insertion of a potassium binding protein, Kbp, from E. coli (Ec-Kbp) into the fluorescent protein mNeonGreen, exhibits an isotonically measured Kd of 69 ± 10 mM (mean ± standard deviation used throughout). We identified Ec-Kbp’s binding site using NMR spectroscopy to detect protein–thallium scalar couplings and refined the structure of Ec-Kbp in its potassium-bound state. Guided by this structure, we modified KRaION1, yielding KRaION1/D9N and KRaION2, which exhibit isotonically measured Kd’s of 138 ± 21 and 96 ± 9 mM. We identified four Ec-Kbp homologues as potassium binding proteins, which yielded indicators with isotonically measured binding affinities in the 39–112 mM range. KRaIONs functioned in HeLa cells, but the Kd values differed from the isotonically measured case. We found that, by tuning the experimental conditions, Kd values could be obtained that were consistent in vitro and in vivo. We thus recommend characterizing potassium indicator Kd in the physiological context of interest before application.

Item Type:Articles
Additional Information:This work was supported by start-up funding from the Foundation of Westlake University, National Natural Science Foundation of China grants 32050410298 and 32171093, 2020 BBRF Young Investigator Grant, and MRIC Funding 103536022023 to K.D.P., by an internal grant of the National Research Center Kurchatov Institute No. 1056 from 02.07.2020 to F.V.S., and by Lisa Yang, John Doerr, and HHMI, and by grants NIH 1R01MH123977, NIH R01DA029639, NIH R01MH122971, NIH R01NS113499, NIH RF1NS113287, NIH RF1DA049005, NSF 1848029, NIH 1R01DA045549, NIH 1R01MH114031, NSF Grant 1734870, NIH R43MH109332, and NIH R01GM104948 to E.S.B. C.C.T.C. was supported by the NSF GRFP Fellowship and Alfred P. Sloan Foundation scholarship.
Status:Published
Refereed:Yes
Glasgow Author(s) Enlighten ID:Smith, Dr Brian
Authors: Torres Cabán, C. C., Yang, M., Lai, C., Yang, L., Subach, F. V., Smith, B. O., Piatkevich, K. D., and Boyden, E. S.
College/School:College of Medical Veterinary and Life Sciences > School of Molecular Biosciences
Journal Name:ACS Sensors
Publisher:American Chemical Society
ISSN:2379-3694
ISSN (Online):2379-3694
Published Online:15 April 2022
Copyright Holders:Copyright © 2022 The Authors
First Published:First published in ACS Sensors 7(5): 1336-1346
Publisher Policy:Reproduced under a Creative Commons License

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