Proceedings of The Physiological Society

University of Oxford (2011) Proc Physiol Soc 23, PC182

Poster Communications

The activation mechanism of rat α3 homomeric Glycine receptors

A. Marabelli1, M. Moroni1, R. Lape1, L. Sivilotti1

1. NPP, University College London, London, United Kingdom.

The α3 subunit of the glycine receptor (GlyR) is found in the spinal cord and hippocampus, where it is thought to be involved in pain sensitivity and in some forms of epilepsy. Despite the physiological relevance of α3-containing channels, there are no studies on kinetic properties of the α3 homomeric receptor. Here we investigated these properties by expressing rat homomeric α3 GlyR (GenBank accession number AJ310838) in HEK293 cells. Cell-attached single channel recordings were obtained at a range of glycine concentrations (50 - 10000 μM). Macroscopic synaptic-like glycine-evoked currents were obtained by rapid applications of brief pulses of saturating glycine (1 ms, 10 mM) to outside-out patches (intracellular chloride concentration 20 mM). Several kinetic mechanisms were tested using maximum likelihood fits (HJCFIT) to the idealized single channel records. The adequacy of each mechanism was judged by comparing the predictions of the model with experimental open/shut time distributions, the single channel open probability curve and the time course of macroscopic glycine-activated currents. We have previously shown that describing the activation of homomeric and heteromeric α1 GlyRs requires models that incorporate a pre-opening intermediate conformational change and reach their maximum open probability when three molecules of glycine have bound (‘flip’, Burzomato et al., 2004). In the present study, this was confirmed for the homomeric α3 GlyR, except that with α3 channels we can see that a fourth molecule of glycine can bind to the channel, something that we could not clarify for the α1 or α1β channel. Open probability was very high both with three and four glycine molecules bound. α1β and α3 GlyRs are similar in their microscopic affinity for glycine and in the efficiency with which the channel flips and then opens. In particular, the opening rate constant was 150,000 ± 20,000 s-1 and the overall efficacy was 62 ± 5 (n = 3 sets; cf. 129,000 s-1 and 20 for α1β heteromers; Burzomato et al., 2004). Again, the microscopic affinity for the agonist glycine was seen to increase as the channel activated, from a resting state value of 1340 ± 200 μM to 180 ± 40 μM (n = 3 sets) for the partially activated flip state. This mechanism and rate constants were also found to describe well the time-course of synaptic-like glycine currents, predicting a decay time constant of 12 ± 4 ms (n = 3 sets), cf. experimental value of 8 ± 3 ms (n = 5 patches; randomisation test, p = 0.20).

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