Both Kir2.1-WT and Kir2.1-R312H mutant were successfully obtained as homogeneous samples in DDM detergent and in amphipols A8-35 (a detergent-free environment) in sufficient quantity to perform structural studies using cryo-electron microscopy (cryo-EM). Two high-resolution structures of the human Kir2.1-WT were successfully determined using cryo-EM, in the absence of PIP2, each in a different conformation. The first structure was obtained using a Kir2.1-WT sample in the presence of DDM detergent. This is the first structure of the human Kir2.1 channel containing both transmembrane domain (TMD) and cytoplasmic domain (CTD). This structure captured the Kir2.1 channel in an extended, closed and non-conductive state The second structure, obtained using a Kir2.1-WT sample in the presence of amphipols A835 (a detergent-free environment), revealed the channel in a compact conformation, but in an inhibited or poorly activated state. Amphipols A8-35 bind to some residues of the PIP2-binding site of Kir2.1 channels in a manner quite similar to how pyrophosphatidic acid (PPA, a competitive inhibitor of Kir2.2 channels) binds to the Kir2.2 structure. Moreover, Kir2.1-WT was successfully reconstituted in MSP:POPC nanodiscs.
KIRPAS was able to provide novel structural data able to describe the molecular mechanisms by which two ATS-causing mutations (C154Y and R312H) affect the function of these channels. Notably, they hinder the channel function by different mechanisms. The cryo-EM structure of the Kir2.1-R312H mutant was obtained at medium resolution; however, molecular modeling analysis revealed that the residue R312 is involved in a network of intersubunit interactions, which are completely disrupted by the R312H mutation and that impairs the channel gating. Molecular dynamics simulations of the Kir2.1-C154Y mutant showed that the C154Y mutation interrupts the structural connection between the flexible extracellular loops and the selectivity filter, resulting in a loss of structural plasticity of the selectivity filter and impairing the K+ flow.
Unfortunately, any cryo-EM structure in the presence of PIP2 was obtained, which impaired to fully describe the structural mechanisms that govern the Kir2.1 gating. However, alternative computational approaches were performed, which revealed that Kir2.1 channel exhibits an inherent compaction motion that PIP2-binding does not induce the compact conformation but rather stabilizes the compressed state. On the other hand, this project was able to provide a high-resolution cryo-EM structure of the Kir2.1 channel bound to with a specific microRNA called m1R1, which was recently discovered as a novel modulator of the Kir2.1 channel function.
The results concerning the cryo-EM structure of Kir2.1-WT channel obtained with samples in DDM were published in Science Advances (Impact Factor: 13.7; doi: 10.1126/sciadv.abq8489). Moreover, the cryo-EM map of Kir2.1-WT (sample in DDM) is publicly available in the Electron Microscopy Data Bank (EMDB) under the code EMD-14678, and the corresponding atomic model is publicly available in the Protein Data Bank (PDB) under the code 7ZDZ. Additionally, the protocol for performing MD simulations are public available at
https://bio-protocol.org/rap30599(opens in new window). These protocols, along with the cryo-EM data processing protocol, were as a book chapter in the book Potassium Channels (Methods in Molecular Biology, volume 2796, doi: 10.1007/978-1-0716-3818-7_10).
The results concerning the cryo-EM structure of Kir-R312H mutant (section 1.2.2.4 above) and the MD simulations of the Kir2.1-C154Y mutant (section 3.1 below) were currently under the second round of peer-reviewing in the Faseb Journal (Impact Factor: 5.9) and are a non-peer-reviewed version is publicly available at bioRxiv (doi: 10.1101/2024.02.09.579451). Moreover, the cryo-EM map of Kir2.1-R312H mutant is publicly available in the EMDB under the code EMD-18595 and its respective atomic model is public available in the Protein Data Bank (PDB) under the code 8QQL.
At least two more papers related to the KIRPAS project are expected to be published in the near future. One will focus on the MD simulations performed in the cryo-EM structure of Kir2.1-WT in amphipols A8-35. The other will cover the cryo-EM structure of Kir2.1-WT bound to m1R1. The results of the KIRPAS project were also presented in conferences and seminars worldwide, including the oral and poster presentation in the 68th Biophysical Society Annual Meeting (Philadelphia, USA) in 2024. Finally, the results of the KIRPAS project were also featured in a report for the general public, produced by the scientific dissemination department from the Sorbonne University:
https://shorturl.at/RnGqN(opens in new window).
