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European Microscopy Congress 2020 Abstract Database

Gating mechanism of a potassium channel, experimental and theoretical studies

Gating mechanism of a potassium channel, experimental and theoretical studies

Session

LSA.11 - CryoEM from membrane proteins to large complexes

Authors

Charline Fagnen (1, 2), Dr Ludovic Bannwarth (1), Dania Zuniga (1), Iman Oubella (1), Dr Said Bendahhou (3), Dr Rita De Zorzi (4), Dr David Perahia (2), Prof. Catherine Vénien-Bryan (1)

Affiliations

1. 1Sorbonne Université, UMR 7590, CNRS, Muséum National d'Histoire Naturelle, Institut de Minéralogie, Physique des Matériaux et Cosmochimie, IMPMC,
2. 2Laboratoire de Biologie et de Pharmacologie Appliquée, Ecole Normale Supérieure Paris-Saclay, Centre National de la Recherche Scientifique,
3. CNRS UMR7370, LP2M, University Nice Sophia-Antipolis, Faculté de Médecine
4. Department of Chemical and Pharmaceutical Sciences, University of Trieste, Via Licio Giorgeri 1, 34127,

Keywords

Biophysical studies,  in silico studies,  Potassium channel, single particle cryo EM 

Abstract text

Summary

Inwardly-rectifying potassium (Kir) channels are transmembrane proteins involved in fundamental physiological processes. Pathologies are directly linked to mutations of this family of channels. For example, some mutations in Kir2.1 cause the Andersen’s syndrome1,2 or hyperinsulinism3 in Kir6.2 channels. This study has three objectives: i) to understand the gating mechanism of the KirBac3.1, ii) to understand the impact of the mutation W46R on the channel, iii) to resolve the structure of the human Kir2.1 at an atomic level.

Introduction

Inwardly-rectifying potassium (Kir) channels are transmembrane proteins responsible for the membrane electrical excitability and K+ transport; it controls the membrane resting potential by opening and closing the K+ channel. Some mutations cause channelopathies4, including Andersen’s syndrome1,2,5, a rare disease due to a loss of function and currently without efficient treatment. This pathology is characterized by periodic paralysis or severe heart problems. On the other hand, some mutations cause a gain of function and favor the opening of the channel. It is the case of Kir6.2, where some mutations are responsible for the hyperinsulenism3. To find the appropriate treatment to treat these pathologies, it is necessary to understand how the molecular mechanism which allows the channel to open and close. This investigation is divided into three parts. The first is led to understand the gating dynamics of the KirBac3.1 channel (a homologous of Kir2.1 and Kir6.2). The goal of the second task is to study the impact of the W46R mutation on the gating. The third objective is to resolve the structure of the human Kir2.1 WT. 

Methods/Materials

The dynamic study was achieved on the wild-type protein KirBac3.1. To study the behavior of this system, Molecular Dynamics using excited Normal Modes (MDeNM)6 method was used. Molecular Dynamics (MD) allows us to observe particularly fast and small amplitude movements such as side-chain or loops movements, while the normal modes describe slow and collective movements of large amplitude. This mixed approach gives access to a wider exploration of the conformational space than MD alone and allows moreover to determine the conformational populations of the different states (open and closed). The simulations carried out with MDeNM provided us with the intermediary structures between the closed and open ones. They allowed us to determine the key motions in the gating such as the involvement of the cytoplasmic domain and slide-helix as well as of the transmembrane helices during the opening of the channel. These observations were confronted with HDX-MS Spectrometry and electrophysiological experiments for validation. Our investigation is continued with some mutations of the channel: KirBac3.1 W46R known to have a higher opening probability compared with the KirBac3.1 WT. To determine the structure of Kir2.1 WT, our laboratory obtained cryogenic electron microscopy images of Kir2.1 from which the determination of its 3D structure is presently being conducted. 

Results and Discussion

The study of the KirBac3.1 dynamics highlights some key motions in the gating of the channel. The analysis of the MDeNM structures shows that the gating of the channel depends on the swing and the rotation of the cytoplasmic domain, the kink of the transmembrane helices, the uprising and the swivel of the slide-helix. This study revealed that the opening at the level of the constriction point Y132 (towards the cytoplasmic domain) was more complex than the one at the level of the second constriction point L124 (middle of the channel).

After the investigation on the mechanism of the channel in the KirBac3.1, we study the impact of the mutation W46R in the KirBac3.1 (W46R in Kir6.2) on the gating mechanism. The comparison between the interaction networks of KirBac3.1 WT and KirBac3.1 W46R revealed that the interactions of the W46 with the inner helix of the channel is disrupted and replaced by interaction with the neighboring slide-helix when the residue is mutated to R46. This modification in the interactions of this residue destabilizes the closed state and facilitate the gating of the channel through a new set of network interaction with the slide helix on the n-1 subunit which triggers the uprising of the slide-helix, which facilitates the opening of the channel. 

Besides these biophysical studies on KirBac3.1 channel, we are focusing now on the structure of the human Kir2.1 potassium channel using cryo-EM combined with image analysis. Preliminary results gave a resolution of the channel at about 7Å. 

Conclusion

These works allow a better understanding of the gating of the Kir family channel and the impact of one of their mutants on the dynamics of the system. The determination of the human Kir2.1 structure is ongoing with promising preliminary results.

References

References

[1] N.M. Plaster et al. Cell 105, 511–519 (2001).

[2] M. Tristani-Firouzi et al. J. Clin. Invest. 110, 381–388 (2002).

[3]  FM. Ashcroft J. Clin. Invest. 115, 2047–2058 (2005).

[4] M.R. Abraham, A  Jahangir, A Terzic, FASEB J. 13, 1901–1910 (1999).

[5] Y Hosaka et al. J. Mol. Cell. Cardiol. 35, 409–415 (2003).

[6] M.G.S. Costa, PR Batista, PM Bisch, D Perahia  J Chem Theory Comput. 2015 11(6):2755-67.