Glycinergic transmission in the spinal cord and brainstem is vital for maintaining skeletal muscle function by inhibiting neuronal excitation. Dysfunctional glycinergic inhibitory transmission underlies the debilitating neurological condition known as hyperekplexia or startle disease. This is characterised by exaggerated startle reflexes, muscle hypertonia and apnoea and its treatment often requires the potentiation, by the use of positive allosteric modulators, of GABA-mediated inhibitory signaling. Here we have investigated the asparagine to lysine (N46K) missense mutation in the glycine receptor (GlyR) α1 subunit gene (GRLA1), which is found in the ethylnitrosourea (ENU) induced murine mutant, Nmf11. The mutation causes a reduced body size, evoked muscle tremor, muscle stiffness, seizures and morbidity usually by postnatal day 21 (Traka et al., 2006). Although N46 lies in close proximity to the glycine binding site, it does not form part of a recognised binding loop or signal transduction pathway. Using whole-cell patch-clamp electrophysiology revealed that introducing the N46K mutation into recombinant GlyR α1 homomeric receptors, expressed in human embryonic kidney (HEK) cells, reduced the agonist potencies of glycine, β-alanine and taurine by 9-, 6- and 3-fold respectively, without significantly affecting their relative maximum responses. In addition, the potency of the competitive GlyR antagonist strychnine was reduced by 15-fold, suggesting that N46K is likely to be affecting binding to the orthosteric binding site rather than agonist efficacy. Replacing N46 with a variety of hydrophobic, charged or polar residues revealed that the amide moiety of asparagine was crucial for GlyR activation. Additionally, from structural modeling studies, co-mutating N61, located on a neighbouring β loop to N46, rescued the wild-type (WT) phenotype depending on the charge of the amino acid side-chain, suggesting that these two residues (N46 and N61) might interact. Single-channel recording of homomeric glycine receptor activity identified that mean burst length for the N46K mutant (3.5 ± 0.46 ms) was reduced when compared with WT receptors (10.4 ± 1.3 ms). Using rapid piezo-driven agonist applications further revealed faster glycine 90 – 10% deactivation/desensitisation times for the N46K mutant (26.1 ± 4.4 ms; n=9) compared to the WT receptor (98.2 ± 10.9 ms; n=10; p<0.05). Values are means ± S.E.M., compared by unpaired t-test. Overall, our data are consistent with N46 ensuring correct alignment of the α1-α1 subunit interface by interaction with juxtaposed residues to preserve the structural integrity of the glycine binding site, which affects the egress of bound glycine from the orthosteric site. This represents a new mechanism by which GlyR dysfunction can induce startle disease.