Investigations of disease-causing mutations to ion channels can often lead to unique insights into the mechanisms of channel function. Normal activation of the glycine receptor (GlyR) is crucial for inhibitory neurotransmission in the brainstem and spinal cord, and one disease that results from impaired GlyR function is human hyperkplexia, or startle disease. Recently, several new hyperekplexia-causing GlyR mutations have been identified and one of these, α1Q226E, exhibits spontaneous activity1. We investigated this mutation because of the potential insights into the mechanisms of channel activation. To this end, we examined the effects of this and nearby mutations on the conductance and kinetics of homo- and heteromeric GlyRs using single-channel and macropatch ensemble recordings from outside-out patches. Our results indicate a higher open probability and longer periods of single channel activity in both saturating and subsaturating glycine concentrations, and a reduced single-channel conductance in α1Q226E-containing GlyRs. Macropatch recordings revealed that synaptic-like events decayed more slowly after agonist removal in α1Q226E GlyRs compared to their wild-type counterparts, indicating a putative increase in the strength of synaptic signaling for α1Q226E-containing GlyRs. We interpret these results to indicate that an interaction between α1Q226E and nearby charged residues influences channel kinetics by stabilizing the open state of the channel. Thus, these results advance our understanding of the mechanisms of hyperekplexia, channel gating and determinants of single-channel conductance in GlyRs.