Bottom-up design of Ca²⁺ channels from defined selectivity filter geometry

Native ion channels play key roles in biological systems, and engineered versions are widely used as chemogenetic tools and in sensing devices^1,2. Protein design has been harnessed to generate pore-containing transmembrane proteins, but the design of selectivity filters with precise arrangements of amino acid side chains specific for a target ion, a crucial feature of native ion channels³, has been constrained by the lack of methods for placing the metal-coordinating residues with atomic-level precision. Here we describe a bottom-up RFdiffusion-based approach to construct Ca²⁺ channels from defined selectivity filter residue geometries, and use this approach to design symmetric oligomeric channels with Ca²⁺ selectivity filters having different coordination numbers and different geometries at the entrance of a wider pore buttressed by multiple transmembrane helices. The designed channel proteins assemble into homogeneous pore-containing particles and, for both tetrameric and hexameric ion-coordinating configurations, patch-clamp experiments show that the designed channels have higher conductances for Ca²⁺ than for Na⁺ and other divalent ions (Sr²⁺ and Mg²⁺) that are eliminated after mutation of selectivity filter residues. Cryogenic electron microscopy indicates that the design method has high accuracy: the structure of the hexameric Ca²⁺ channel is nearly identical to that of the design model. Our bottom-up design approach now enables the testing of hypotheses relating filter geometry to ion selectivity by direct construction, and provides a roadmap for creating selective ion channels for a wide range of applications.