Mammalian auditory amplifier as a voltage-sensing core for the genetically encoded indicators

Session

LSA.7 - Pathology, immunocytochemistry and biomolecular labelling

Authors

Mrs Liubov Kost (2), Mrs Violetta Ivanova (1), Ms Valentina Yunusova (2), PhD DrSc Evgeny Nikitin (1), PhD DrSc Konstantin Lukyanov (2, 3), PhD Alexey Bogdanov (2)

Affiliations

1. Institute of Higher Nervous Activity of RAS
2. Shemyakin-Ovchinnikov Institute of bioorganic chemistry
3. Skolkovo Institute of Science and Technology

Keywords

auditory analyzer, fluorescent proteins, genetically encoded voltage indicator, membrane potential, prestin

Abstract text

Prestin is a voltage-sensitive motor protein specifically expressed in the outer hair cells and providing auditory signal amplification in the mammalian cochlea due to the voltage-dependent conformational changes [1]. Its extremely fast voltage-dependent conformational dynamics and a significant mechanical force produced by its molecule, as well as a native expression in the nerve cells of higher animals make it a promising candidate for a voltage sensing domain in the genetically encoded voltage indicators (GEVIs).

In this contribution, we aimed to prove prestin’s ability to function as the GEVIs sensitive core. To that end, we have designed several genetic constructs based on Meriones unguiculatus prestin [1] and FusionRed fluorescent protein [2]. Since little is known about the molecular mechanism of prestin electromotility and therefore there are no ready-to-use principles for prestin-based indicators designing, we have suggested 3 diverse topologies of the indicator molecules. They include “insertion-into-cpFP” (circular permuted Fluorescent Protein)  that we implemented earlier for Ciona Intestinalis voltage sensitive domain-based GEVIs [3,4], “insertion-into-FP” that has been widely used in fluorescent indicators development, and “cpFP insertion-into-prestin” that could become a perspective approach applied to the large transmembrane proteins.

Our whole-cell voltage-clamp experiments with live HEK293T cells revealed that the prestin-based GEVI (at least built on “insertion-into-cpFP” topology) demonstrates fast (submillisecond) fluorescent response to the membrane potential changes. Apart from the proof-of-principle of prestin applicability in GEVIs, we have shown that the cpFusionRed insertions into prestin intracellular/external loops often leads to the altered fusion trafficking and/or aggregation [5].

The authors gratefully acknowledge funding RFBR according to the research projects № 18-34-20087 and 19-34-90140, Electrophysiology experiments were supported by RFBR grant 19-015-00022. Experiments were partially carried out using the equipment provided by the IBCh core facility (CKP IBCh, supported by Russian Ministry of Education and Science, grant RFMEFI62117X0018).

References

1. Zheng J, Shen W, He D, et al. Prestin is the motor protein of cochlear outer hair cells. Nature 405 (2000), 149–155.

2. Shemiakina II, Ermakova, GV, Cranfill, PJ, et al. A monomeric red fluorescent protein with low cytotoxicity. Nat Commun 3 (2012), 1204.

3. Kost LA, Nikitin ES, Ivanova VO, et al. Insertion of the voltage-sensitive domain into circularly permuted red fluorescent protein as a design for genetically encoded voltage sensor. PLOS ONE. 12(9) (2017),

4. Kost, LA, Ivanova VO, Balaban PM, et al. Red Fluorescent Genetically Encoded Voltage Indicators with Millisecond Responsiveness. Sensors. 19 (13) (2019.), 2982.