Dynamic dimer-of-dimers architecture defines Mg²⁺ transport in human CNNM4

Mg²⁺ is essential for all living organisms, yet its transport across mammalian membranes remains poorly understood. Here, we present cryoelectron microscopy (cryo-EM) structures of a full-length mammalian Mg²⁺ transporter on the plasma membrane, human CNNM4, in outward-facing and occluded states, revealing an unexpected tetrameric assembly organized as a dimer of asymmetric dimers-distinct from the symmetric dimers in prokaryotic homologs and long assumed for eukaryotic CNNMs. We show that Mg²⁺/ATP binding stabilizes the dynamic intracellular domains and promotes tetramerization, while an acidic patch binds additional Mg²⁺, potentially acting as a sensor to couple cytoplasmic Mg²⁺ levels to transport activity. Within the transmembrane domain, a key glutamate flips upon Na⁺ binding and destabilizes the Mg²⁺-binding site in the outward-facing state, thereby promoting Mg²⁺/Na⁺ exchange. Together, these findings establish a mechanistic framework for CNNM transport and regulation that diverges from prokaryotic models and links CNNM function to human physiology and disease.