Startle disease is a rare neurological disorder, producing an exaggerated startle response to auditory, visual and tactile stimuli. It is due to a deficiency in glycinergic neurotransmission, most often caused by mutations in the glycine receptor (GlyR). This study describes the functional consequences of a novel startle mutation, W170S, in the alpha1 subunit of the human GlyR. Using a combination of homology modelling with mutagenesis and electrophysiology studies, we have investigated the functional changes at the whole-cell and single channel level. We propose that the W170 residue normally forms a H-bond that links the inner and outer β-sheets of the extracellular domain, contributing to the stability the receptor conformation when ligand is bound.