Phosphorylation of Serine 263 in the Human Choline Transporter 1 alters the transporter's function

Disruptions of the neuromuscular junction result in congenital myasthenic syndromes (CMS). CMS is characterized by akinesia, apneas or even death. CMS type 20 is caused by mutations in the SLC5A7 gene which encodes for the choline transporter (CHT1). The 13 transmembrane domain CHT1 protein is thought to be phosphorylated at several sites but the role of phosphorylation remains unknown. We have recently reported the missense recessive substitution c.C788>T in SLC5A7 in a family with a history of CMS using whole exome sequencing. This mutation encodes a p.S263F substitution in CHT1 and results in a complete loss of transport function despite a normal plasma membrane localization. To assess whether 1) the p.S263F substitution alters CHT1 phosphorylation state, and 2) a change in phosphorylation results in altered transport function, three substitutions were introduced into CHT1: p.S263A, and phosphomimetic p.S263D and p.S263E. Upon expression in HEK293 cells, we found that p.S263A mutant retained a high level of transport activity while the p.S263D and p.S263E mutants were non‐functional, suggesting that S263 phosphorylation inhibits choline transport. Immobilized metal affinity electrophoresis supports that wild‐type CHT1 may be more phosphorylated than p.S263F CHT1 mutant. These results support the essential role of Serine 263 residue in CHT1 choline transport possibly via altering its phosphorylation status. Support or Funding Information Women and Children’s Health Research Institute, Stollery Children’s Hospital Foundation, Canadian Institute of Health Research & The University of Alberta