From extracellular entry to intracellular release: A water-assisted transport cycle for creatine in SLC6A8.

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.