Mutations in genes encoding solute carriers are frequently associated with human diseases, yet often the molecular basis of how these mutations disrupt the corresponding transport remains unclear. The creatine transporter (CRT), encoded by the X-linked SLC6A8 gene, is one of such cases and is responsible for creatine uptake into energy-demanding tissues such as muscle, heart, and brain. Dysfunction of CRT caused by pathogenic variants leads to creatine transporter deficiency (CTD), a disorder characterized by intellectual disability, hypotonia, seizures, and speech delay. Although CRT belongs to the SLC6 family and operates via an alternating access mechanism, the detailed transport pathway and its alteration by disease mutations remain unresolved. Classical molecular dynamics fall short of efficiently exploring the large conformational changes required for alternating access. To address this, we applied constant-force steered molecular dynamics (SMD) together with targeted MD (tMD) to simulate the complete transport cycle, from the outward-open to the inward-open state. Our simulations of the wild-type transporter reveal a consistent creatine translocation pathway, influenced by ions and coupled to both global and local conformational changes in the protein. These results agree with previous work and are consistent with the alternating access mechanism described for other SLC6 transporters. Building on this framework, we examined pathogenic variants and, for the first time, simulated their full transport pathway. We find that mutations perturb key conformational transitions and substrate-coordination residues, providing mechanistic insight into how these variants impair transport and lead to CTD. This study provides the first molecular-level simulation of the complete transport cycle in both wild-type and mutant CRT, laying the groundwork for understanding the structural basis of disease-causing mutations.
Pitambar Poudel (Sun,) studied this question.