From extracellular entry to intracellular release: A water‐assisted transport cycle for creatine in <scp>SLC6A8</scp>

Abstract The creatine transporter (CRT/SLC6A8) plays a key role in cellular energy homeostasis, yet the molecular mechanism underlying creatine transport remains poorly understood. Here, we reconstruct the complete transport cycle of human CRT using a hybrid simulation strategy that combines constant‐force steered molecular dynamics (cf‐sMD) with targeted molecular dynamics (tMD). This approach captures continuous progression through the outward‐open, outward‐occluded, inward‐occluded, and inward‐open states and reveals a water‐assisted, sequential intracellular release of Na2, creatine, and Na1. Hydration analysis shows that progressive water penetration into the binding pocket weakens protein‐substrate and protein‐ion interactions and destabilizes the bound state before release. Residue‐level contact analysis identifies residues that interact with creatine along the transport pathway, while dynamic network analysis reveals a TM1–TM6 communication backbone that mediates long‐range coupling during transport. Together, these results provide a molecular framework for creatine transport and establish an approach for investigating transport mechanisms across the broader solute carrier family.