A brief guideline for the studies of structure-function relationship of ion channels using AlphaFold3.

Ion channels are crucial membrane proteins that regulate ion flux, thus affecting a broad range of physiological processes and disease mechanisms. Traditional approaches, such as X-ray crystallography and cryo-electron microscopy, often face significant challenges in resolving the structure-function relationships of ion channels due to the complex subunit assemblies, transient functional states, and high experimental costs. AlphaFold3 offers a major leap forward by accurately modeling multimeric channels, predicting ligand and ion interactions, and refining subunit interfaces. Building on AlphaFold2, it integrates diffusion-based algorithms and enhanced confidence metrics to explore gating, auxiliary subunits, disease-linked mutations, etc. This review outlines AlphaFold3's key innovations and provides a step-by-step protocol for its use in predicting ion channel complexes. We discuss essential structural parameters, common software for basic analyses, and complementary techniques including molecular dynamics simulations, molecular docking, functional assays (electrophysiology and ion imaging) and structure biology techniques that extend and validate computational findings. Representative examples, including the P2X receptors and voltage-gated calcium channels, demonstrate AlphaFold3's ability to clarify channel assembly, gating mechanisms, channelopathies, and therapeutic opportunities. Finally, we address AlphaFold3's remaining limitations. Despite these hurdles, AlphaFold3 offers a transformative platform that, when integrated with established experimental methods, is poised to significantly advance ion channel research and drug discovery.