Kinetic properties of the α₂ homo‐oligomeric glycine receptor impairs a proper synaptic functioning

Abstract

Ionotropic glycine receptors (GlyRs) are present in the central nervous system well before the establishment of synaptic contacts. Immature nerve cells are known, at least in the spinal cord, to express α2 homomeric GlyRs, the properties of which are relatively unknown compared to those of the adult synaptic form of the GlyR (mainly α1/β heteromeres). Here, the kinetics properties of GlyRs at the single‐channel level have been recorded in real‐time by means of the patch‐clamp technique in the outside‐out configuration coupled with an ultra‐fast flow application system (< 100 µs). Recordings were performed on chinese hamster ovary (CHO) cells stably transfected with the α2 GlyR subunit. We show that the onset, the relaxation and the desensitisation of α2 homomeric GlyR‐mediated currents are slower by one or two orders of magnitude compared to synaptic mature GlyRs and to other ligand‐gated ionotropic channels involved in fast synaptic transmission. First latency analysis performed on single GlyR channels revealed that their slow activation time course was due to delayed openings. When synaptic release of glycine was mimicked (1 mm glycine; 1 ms pulse duration), the opening probability of α2 homomeric GlyRs was low (Po≈ 0.1) when compared to mature synaptic GlyRs (Po= 0.9). This low Po is likely to be a direct consequence of the relatively slow activation kinetics of α2 homomeric GlyRs when compared to the activation kinetics of mature α1/β GlyRs. Such slow kinetics suggest that embryonic α2 homomeric GlyRs cannot be activated by fast neurotransmitter release at mature synapses but rather could be suited for a non‐synaptic paracrine‐like release of agonist, which is known to occur in the embryo.

Inhibitory Control at a Synaptic Relay

“…Together, these properties of glycinergic transmission suggest that it is well suited for modifying excitation with temporal precision. Given that IPSC decay kinetics at room temperature also exceeded those reported for receptors in patches excised from neurons (Singer and Berger, 1999;Ali et al, 2000) or from cells expressing human or zebrafish ␣1 subunits (Fucile et al, 1999;Gentet and Clements, 2002) or ␣2 subunits (Mangin et al, 2003), it seems likely that the receptors in mature MNTB contain a novel subunit or posttranslational modification.…”

Glycine Release from Radial Cells Modulates the Spontaneous Activity and Its Propagation during Early Spinal Cord Development

“…To make a "sniffer" electrode (Young and Poo, 1983), an outside-out patch was pulled from a transfected CHO cell line expressing the GlyR ␣2 subunit (Mangin et al, 2003). Homomeric GlyR ␣2 subunits, highly specific for glycine, were characterized by a high elementary conductance (100 -120 pS), a long mean open time, and little desensitization, making them a good sensor for glycine (Mangin et al, 2003). As a control, the electrode was first positioned outside the SC to verify any glycine contamination in the recording medium.…”

“…For electrophysiological recordings, cells were seeded onto glass coverslips coated with poly-L-ornithine (0.1 mg/ml). Glycine receptor ␣ 2 subunit cloning and transfection were performed as described by Mangin et al (2003).…”

Single-Channel Behavior of Heteromeric α1β Glycine Receptors: An Attempt to Detect a Conformational Change before the Channel Opens

“…These problems will be the subject of future work, but preliminary results suggest that experiments simulated with the flip mechanism can be fitted very well with scheme 3 (18 free parameters) and that results simulated with scheme 3 can be fitted with the flip mechanism (14 free parameters) in a way that is not perfect but is sufficiently good that it would not be distinguishable in practice, with the sort of recordings that we have made. Possible ways of distinguishing between the rival mechanisms include looking at a range of agonists (in progress), recording single channels after a concentration jump, increasing resolution by using ultra-low noise methods, decreasing temperature, or using the slow ␣2 subunit (Mangin et al, 2003).…”