Cryo-electron microscopy structures of human Kir2.1 potassium channel and molecular dynamics simulations provide insight into its gating mechanism and the impact of disease-related mutants

Kir2.1 channels are inward-rectifying potassium channels essential for maintaining the resting membrane potential in excitable cells. Genetic mutations in Kir2.1 have been associated with several rare human diseases, notably Andersen’s syndrome (AS)—a neuromuscular disorder for which current treatments are largely ineffective. The gating of Kir2.1 is regulated by the phospholipid phosphatidylinositol-4,5-bisphosphate (PIP₂), which is essential for channel activation and physiological function. Here, we present the cryo-EM structures of human Kir2.1 channels in two states: the non-conductive, closed (apo) form (at 4.3 Å resolution), and the PIP₂-bound form (at 2.8 Å resolution). The apo structure provides valuable insights into the multiple conformational states of the channel and underscores the role of the G-loop, located in the cytoplasmic domain, in regulating pore aperture. Preliminary analysis of the Kir2.1/PIP₂ complex reveals that the PIP₂-binding site lies within a positively charged region, enriched in lysine and arginine residues, at the interface between the transmembrane and cytoplasmic domains. Structural comparisons between the apo and PIP2-bound forms demonstrate that PIP₂ binding induces significant conformational rearrangements, including a reorganization of the Glu303 interaction network—changes that collectively lead to pore opening at the G-loop level. Additionally, cryo-EM data combined with molecular dynamics simulations of the AS-associated mutations R312H and C154Y, which do not affect PIP₂ binding, revealed distinct mechanisms of channel dysfunction. The R312H mutation impairs gating at the G-loop, whereas C154Y affects K⁺ conduction at the selectivity filter. Our data support a dominant-negative effect of C154Y, where a single mutated subunit within the tetramer is sufficient to render the entire channel non-functional. These findings shed light on the gating mechanism of Kir2.1 and enhance our understanding of the molecular basis by which AS-associated mutations impair channel function.

This work was supported by European Union Horizon Europe Research and innovation Program under grant agreement no. 101026386.