Specific residues in the CNNMs regulate Mg ²⁺ transport function

Cyclin M (CNNM) is a family of magnesium (Mg 2+ ) transporters, consisting of four members with distinct tissue expression in mammals. Previous studies have postulated roles of CNNM proteins in Mg 2+ uptake, sodium dependent Mg 2+ extrusion and regulation of other Mg 2+ channels. However, the functional differences between CNNM1-4 have never been systematically assessed. The aim of this study is to examine the Mg 2+ transport properties of all four CNNM proteins and confirm differential function between family members as well as the function of specific residues in Mg 2+ transport function. To further understand the protein complex composition, we aim to identify interaction partners of CNNM2. 25Mg extrusion assays were conducted in HEK293 cells knock-out for CNNM3/CNNM4 and transfected with individual constructs for each CNNM family member. CNNM2 and CNNM4 over expressing cells had an approximately 2-fold higher Mg 2+ extrusion compared to mock (p< 0.05). To further map the functional properties of CNNMs, the discriminative amino acids in the putative Mg 2+ pore of CNNM3 were mutated into the amino acids specific for the respective putative Mg 2+ pore of CNNM4 and vice versa. Structural modelling of the WT CNNM proteins as well as proteins with mutated pore regions showed that the introduced mutations do not lead to major structural changes of the proteins. This suggests that the differences in Mg 2+ transport capability is linked to the Mg 2+ binding pocket. Mutation of the putative Mg 2+ binding sites of CNNM4 into CNNM3 resulted in 180% higher Mg 2+ extrusion compared to the original CNNM3, while mutation of the putative Mg 2+ binding sites of CNNM3 into CNNM4 resulted in 22% lower Mg 2+ extrusion compared to CNNM4. Proximity biotinylation assays were performed and subsequently analysed using Mass spectrometry to identify new interaction partners of the CNNM2. CNNM2 binds to transient receptor potential cation channel 7 (TRPM7) at the membrane and complex formation depends on Mg 2+ concentrations. In conclusion, our results show that CNNM2 and CNNM4 facilitate Mg 2+ extrusion, in contrast to CNNM1 and CNNM3. We propose that specific residues in the CNNM4 pore create the unique physical properties required for the extrusion of Mg 2+ . We also show that a CNNM2-TRPM7 complex is formed at the cellular membrane and is dependent on Mg 2+ concentrations. This abstract was presented at the American Physiology Summit 2026 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.