Abstract
Glycine receptors mediate inhibitory synaptic currents that are essential for motor control. Loss-of-function mutations in human glycine receptors cause hyperekplexia, a rare inherited disease associated with an exaggerated startle response. We have studied a human disease mutation in the M2-M3 loop of the glycine receptor alpha1 subunit (K276E) using direct fitting of mechanisms to single-channel recordings with the program HJCFIT. Whole-cell recordings from HEK 293 cells suggested that mutant receptors, both homomeric alpha1 and heteromeric alpha1beta, were much less sensitive to glycine than their wild-type counterparts. Single-channel recordings showed that homomeric alpha1 K276E receptors are barely active, even at 200 mM glycine. In contrast, heteromeric channels show brief bursts of openings at 300 µM glycine (∼EC95 for wild-type) and reach a maximum one-channel open probability of about 45% at 100 mM glycine (cf. 96% for wild-type). Distributions of apparent open times contained more than one component at high concentrations, and so mechanisms that allowed only one open state in the fully liganded receptor were extremely poor at describing the observed single-channel behaviour. Instead, mechanisms in which opening can also occur from more than one fully liganded intermediate state (e.g. ‘primed’ models) gave a much better description of the data. Brief pulses of glycine (0.5-2 ms, 30 mM) applied to outside-out patches activated currents with a slower rise time (1.3 ms) than wild-type channels (0.2 ms), and a much faster decay. These features of the macroscopic currents were well predicted by the mechanisms obtained from fitting single-channel data. Our results show that the mutation K276E impairs the gating of the receptor, demonstrate an unforeseen role for the beta subunit in promoting channel opening, and give further evidence of reaction intermediates in the activation pathway.
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