The glycine receptor (GlyR) is a ligand-gated chloride channel and a member of the Cys-loop superfamily. It is responsible for some of the fast synaptic inhibition in brain and spinal cord. Heritable malfunction of glycinergic transmission in man causes hyperekplexia, a neuromotor disorder characterised by exaggerated startle responses to normal sensory stimuli. Many mutations responsible for the disease are found in GlyR subunits, where they highlight residues essential for channel activation.We evaluated the effects of three human hyperekplexia α1 subunit mutations, S231N, Q266H and S267N, on human heteromeric α1β GlyR expressed in HEK293 cells. Glycine dose-response curves obtained by whole-cell patch-clamp (holding potential – 50 mV) confirmed previous reports (Bode & Lynch, 2014) that these mutations decrease the channel sensitivity to glycine. Thus, glycine EC50 increased from its wild type value of 0.33 ± 0.1 mM (n = 5), to 3.8 ± 0.4 (n = 4), 3.5 ± 0.5 (n = 5) and 1.4 ± 0.1 mM (n = 3) for S231N, S267N and Q266H, respectively (mean ± S.E.M). In order to understand the mechanism of action of the mutations, we performed also single-channel recordings (cell-attached, pipette potential +100 mV) at saturating glycine concentration. This allowed us to measure the channel maximum open probability (Popen = cluster open time / total cluster time). Wild type GlyRs open with very high maximum Popen (0.98 ± 0.01, n = 18 clusters), whereas clusters recorded from mutant receptors had much lower maximum Popen, 0.46 ± 0.05 (n = 5), 0.55 ± 0.07 (n = 9) and 0.41 ± 0.03 (n = 4) for S231N, Q266H and S267N, respectively. This reduction in maximum Popen was clear, despite the presence of distinct gating modes (stretches of activations with different Popen) in mutant receptors. These data suggest that the human hyperekplexia mutations tested here shift glycine EC50 by reducing gating efficacy.