Structural and dynamic characterization of a human potassium channel (Kir) in normal and pathological states – KirFlex

Cells employ allosteric regulation as a fundamental mechanism to control critical processes at the molecular level. Computational approaches based on molecular simulations enable in-depth, comparative analyses of allosteric regulation networks, especially when combined with cryo-EM data and biophysical methods such as single molecule FRET (smFRET).
The inward rectifier potassium (Kir) channels control the permeation of K+ ions. They are ubiquitously expressed throughout the human body and are important regulators of electrical signaling in the brain, muscle, pancreas, liver, and cardiovascular system. They are also important regulators of salt balance in the kidney. The gating of Kir2.1 is modulated by modulators such as PIP2, anionic lipids, microRNAs (miR). Mutations on Kir2.1 cause several disorders in humans such as Andersen Syndrom (AS).
The main objective of KirFlex is to understand the dynamical rearrangement of Kir channels and the allosteric regulation pathways during gating. We will investigate the functional effect of regulators such as PIP2, anionic lipids and miR1 in interaction with Kir2.1 and the impact of two medically important, potentially allosteric Kir2.1 channel mutants: R312H, a loss of function mutant, and M301K, a gain of function mutant. Despite the considerable advances in the biochemical knowledge of the Kir channels, the detailed mechanism by which Kir channels are regulated remains unclear. How mutants cause gating defects is also not known.
This project has interdisciplinary experimental and theoretical innovative approaches by the integration of cutting-edge structural (cryo-EM-SPA) and biophysical techniques (smFRET in order to investigate the dynamics of the channel and Surface Plasmon Resonance -SPR). In order to access the many transition states, (which are difficult to access experimentally) we will use in silico methods, particularly a computational methodologies (MDeNM) method recently developed by partner 2.
At the conclusion of this project, the structure at high resolution of the human Kir2.1 WT and mutants in complex with modulators such as PIP2, negatively charged lipids, miR1 will be deciphered. The allosteric regulation pathways involved in the gating of the channel and the understanding of the impact of clinically-relevant disease-causing mutations on the structure, dynamics, and function of Kir channels will be unveiled, laying the foundations for future investigation of rationally designed allosteric modulator treatments targeting Kir channels pand articularly developing an innovative class of RNA medicines to treat muscular, cardiac, and bone formation defects associated with AS and other ion channel disorders.
The work package of KirFlex are
i)..To determine the high-resolution structures of the human Kir2.1 WT and disease-related mutants in complex with PIP2, negatively charged phospholipids, and a specific miR1.
ii) To investigate the function and dynamics of these channels (WT and mutants) using smFRET and SPR.
iii) To use computational methods to interpret and integrate the experimental results to decipher the allosteric regulation pathways involved in the channel's gating and understand the molecular mechanism of mutant dysfunction.
The transdisciplinary consortium assembles two complementary teams with demonstrated expertise in the structure and function of ion channels (Vénien-Bryan C, IMPMC- Sorbonne Université Paris partner 1), and in the integration of experiments and computational methods to study macromolecular dynamics (Pasi M, ENS Paris-Saclay, partner 2). The project is ambitious but remains realistic, considering the partners' expertise and the preliminary results already at hand.