Pentameric ligand-gated ion channels (pLGICs) mediate numerous physiological processes and are therapeutic targets for a wide range of clinical indications. Elucidating the structural differences between their closed and open states may help in designing improved drugs that bias receptors towards the desired conformational state. Human hereditary hyperekplexia (or startle disease) is most commonly caused by missense or nonsense mutations that disrupt the function of the glycine receptor alpha1 subunit. We recently showed that two new hyperekplexia mutations, Q226E and V280M, induced spontaneous activity in homomeric alpha1 glycine receptors. We reasoned that understanding the molecular mechanisms by which these mutations produce tonic activation may reveal how glycine receptors activate. Q226, located near the top of TM1, is closely apposed to R271 at the 19' position at the top of TM2 in the neighbouring subunit. Using mutant cycle analysis we inferred that Q226E induces activation via an enhanced electrostatic attraction to R271. This would tilt the top of TM2 towards TM1 and hence away from the pore axis to open the channel. We also concluded that the increased side chain volume of V280M, in the TM2-TM3 loop, exerts a steric repulsion against I225 at the top of TM1 in the neighbouring subunit. We infer that this steric repulsion would tilt the top of TM3 radially outwards against the stationary TM1 and thus provide space for TM2 to relax away from the pore axis to create an open channel. Because the transmembrane domain movements inferred from this functional analysis are consistent with the structural differences evident in the X-ray atomic structures of closed and open state bacterial pLGICs, we propose that the model of pLGIC activation as outlined here may be broadly applicable across the eukaryotic pLGIC receptor family.