Transport of the clinically relevant creatine transporter (SLC6A8) to the cell membrane may depend upon five C-terminal amino acids

Creatine is essential for energy storage and distribution within human cells. The creatine transporter (SLC6A8) mediates cellular uptake of creatine. Mutations have profound clinical consequences. Transport of creatine transporter from ER to cell membrane modulates creatine uptake activity and is required for function. Comparison with related SLC6 transporters suggested C-terminal motifs are required for movement of the creatine transporter to the cell membrane. Plasmids encoding C-terminal truncation variants of creatine transporter were created and expressed in HEK293 cells. Western blotting biotinylated cell surface protein suggested that creatine transporter truncation variants of up to 35 amino acids reached the cell membrane, whilst truncations of 40 or more were retained within the cell. 14 C-Creatine uptake assays demonstrated variants truncated by up to 35 amino acids were functional in the uptake of creatine, whilst truncations of 40 or greater were not. The intervening five C-terminal amino acids (QPIWG) may be required for creatine transporter movement to the cell membrane, and for function. These amino acids include a triplet of hydrophobic amino acids. A similarly positioned triplet of hydrophobic amino acids (VMI) appears required for the transport of related GAT1 from the ER-Golgi intermediate complex to the cell membrane. A comparably positioned triplet of hydrophobic amino acids is present in other SLC6 family members. Experimentation with other SLC6 family members is needed to further examine these findings.