Alexandra Kitzenmaier, Natascha Schaefer, Vikram Babu Kasaragod, Tilman Polster, Ralph Hantschmann, Hermann Schindelin, Carmen Villmann

• Glycine transporter 2 (GlyT2) mutations across the entire sequence have been shown to represent the presynaptic component of the neurological disease hyperekplexia. Dominant, recessive and compound heterozygous mutations have been identified, most of them leading to impaired glycine uptake. Here, we identified a novel loss of function mutation of the GlyT2 resulting from an amino acid exchange of proline 429 to leucine in a family with both parents being heterozygous carriers. A homozygous child suffered from severe neuromotor deficits. WeGlycine transporter 2 (GlyT2) mutations across the entire sequence have been shown to represent the presynaptic component of the neurological disease hyperekplexia. Dominant, recessive and compound heterozygous mutations have been identified, most of them leading to impaired glycine uptake. Here, we identified a novel loss of function mutation of the GlyT2 resulting from an amino acid exchange of proline 429 to leucine in a family with both parents being heterozygous carriers. A homozygous child suffered from severe neuromotor deficits. We characterised the GlyT2P429L variant at the molecular, cellular and protein level. Functionality was determined by glycine uptake assays. Homology modelling revealed that the mutation localises to α‐helix 5, presumably disrupting the integrity of this α‐helix. GlyT2P429L shows protein trafficking through various intracellular compartments to the cellular surface. However, the protein expression at the whole cell level was significantly reduced. Although present at the cellular surface, GlyT2P429L demonstrated a loss of protein function. Coexpression of the mutant with the wild‐type protein, reflecting the situation in the parents, did not affect transporter function, thus explaining their non‐symptomatic phenotype. Nevertheless, when the mutant was expressed in excess compared with the wild‐type protein, glycine uptake was significantly reduced. Thus, these data demonstrate that the proline residue at position 429 is structurally important for the correct formation of α‐helix 5. The failure in functionality of the mutated GlyT2 is most probably due to structural changes localised in close proximity to the sodium‐binding site of the transporter.…