A Reluctant Gating Mode of Glycine Receptor Channels Determines the Time Course of Inhibitory Miniature Synaptic Events in Zebrafish Hindbrain Neurons

Abstract

Miniature IPSCs (mIPSCs) recorded in the Mauthner (M)-cell of zebrafish larvae have a broad amplitude distribution that is attributable only partly to the functional heterogeneity of postsynaptic glycine receptors (GlyRs). The role of the kinetic properties of GlyRs in amplitude fluctuation was investigated using fast-flow application techniques on outside-out patches. Short applications of a saturating glycine concentration evoked outside-out currents with a biphasic deactivation phase as observed for mIPSCs, and they were consistent with a rapid clearance of glycine from the synaptic cleft. Patch currents declined slowly during continuous applications of 3 mM glycine, but the biphasic deactivation phase of mIPSCs cannot reflect a desensitization process because paired-pulse desensitization was not observed. The maximum open probability (Po) of GlyRs was close to 0.9 with 3 mM glycine. Analyses of the onset of outside-out currents evoked by 0.1 mM glycine are consistent with the presence of two equivalent binding sites with a Kd of O.3-O.4 mM. Activation and deactivation properties of GlyRs were better described with a kinetic model, including two binding states, a doubly liganded open state, and a reluctant gating mode leading to another open state. The 20-80% rise time of mIPSCs was independent of their amplitude and is identical to that of outside-out currents evoked by the applications of a saturating concentration of glycine (>1 mM). These results support the hypothesis that GlyR kinetics determines the time course of synaptic events at M-cell inhibitory synapses and that large mIPSC amplitude fluctuations are mainly of postsynaptic origin.

Glycine Receptors in CNS Neurons as a Target for Nonretrograde Action of Cannabinoids

“…Slow desensitization of GlyRs cannot play a significant role after release of a single vesicle. However, it might contribute to glycinergic synaptic efficacy when a longlasting (seconds-range) depolarization of the postsynaptic membrane is coupled with high-frequency cell activity (Legendre, 1998). Moreover, the prolonged deactivation phase of glycinergic events should act to reinforce the efficacy of inhibition.…”

Mechanisms for Picrotoxinin and Picrotin Blocks of α2 Homomeric Glycine Receptors

“…Drug Delivery-Outside-out single channel currents were evoked using a fast-flow operating system (22,23). Control and drug solution were gravity-fed into two channels of a thin-wall glass theta tube (2 mm outer diameter; Hilgenberg, Malsfeld, Germany), pulled, and broken to obtain a 200-m tip diameter.…”

“…The solution exchange was performed by rapidly moving the solution interface across the tip of the patch pipette, using a piezo-electric translator (model P245.30, Physik Instrument, Waldbronn, Germany). Concentration steps of glycine lasting 1-1000 ms were applied with an interval of Ն10 s. Exchange time of 10 -90% (Ͻ100 s) was determined before each set of experiments by monitoring the change in the liquid junction potential evoked by the application of a 10% diluted control solution to the open tip of the patch pipette (23). For the experiments requiring fast solution exchange between three different conditions (see Figs.…”

“…Decay time constants were obtained by fitting the first 750 ms of the decay phase with a sum of exponential curves using Axograph 4.9 software (Axon Instruments). The presence of one or more exponential components was tested by comparing the sums of squared errors (SSEs) of the fits (23,24).…”

The Activation Mechanism of α1 Homomeric Glycine Receptors

“…Indeed, it is almost impossible to estimate all of the rate constants (in a model in which the two sites are initially different), unless the assumption that sites do not interact is made (Colquhoun et al, 2003a). Similar assumptions have been made for GABA A and glycine receptors (Jones and Westbrook, 1995;Legendre, 1998).…”