The kinetics of inhibition of rat recombinant heteromeric α1β glycine receptors by the low‐affinity antagonist SR‐95531

Abstract

The GABA A antagonist SR-95531 (gabazine) is known to block glycine receptors, albeit with low affinity. We have studied the effect of SR-95531 on rat recombinant α1β glycine receptors expressed in human embryonic kidney (HEK293) cells by recording macroscopic currents elicited by rapid glycine application to outside-out patches. SR-95531 has a fast unbinding rate (k offSR , about 3000 s −1 ) and this means that the time course of its unbinding is comparable to the expected time course of the transmitter in the cleft. We also found that equilibrium applications of SR-95531 reduced the response to brief glycine applications by an amount inversely proportional to the duration of glycine application. The fast unbinding rate of SR-95531 from the glycine receptor will make it useful for establishing the time course of glycine concentration at glycinergic synapses.

Rapid, Activity-Independent Turnover of Vesicular Transmitter Content at a Mixed Glycine/GABA Synapse

“…We tested for such saturation by measuring the degree of block of IPSCs by the low-affinity, competitive glycine receptor antagonist SR95531, under basal conditions and in pairs with 20 mM glycine in the presynaptic pipette. SR95531 blocks GABA A receptors with high affinity, although it also acts as a low-affinity competitive antagonist of glycine receptors at concentrations >30 μM (Beato et al, 2007; Beato, 2008). The off-rate of SR95531 from glycine receptors (~300 μs) is shorter than the duration of the synaptic glycine transient (Beato et al, 2007).…”

“…SR95531 blocks GABA A receptors with high affinity, although it also acts as a low-affinity competitive antagonist of glycine receptors at concentrations >30 μM (Beato et al, 2007; Beato, 2008). The off-rate of SR95531 from glycine receptors (~300 μs) is shorter than the duration of the synaptic glycine transient (Beato et al, 2007). Thus, a significant fraction of glycine receptors will unbind SR95531 while glycine is still present in the synaptic cleft, making the degree of block by a set concentration of SR95531 inversely proportional to the amount of glycine released in the cleft following exocytosis (Beato, 2008).…”

The Glycine Transporter GlyT2 Controls the Dynamics of Synaptic Vesicle Refilling in Inhibitory Spinal Cord Neurons

“…5B), we examined whether there was a presynaptic change in glycine release by taking advantage of SR95531, a low-affinity competitive antagonist of GlyRs (Wang and Slaughter, 2005;Beato et al, 2007). Inhibition of GlyRs by a competitive antagonist should be sensitive to changes in the amount of glycine released (Clements, 1996) because SR95531 has an estimated mean lifetime occupancy of 300 s and should unbind within the timescale of synaptic release (Beato et al, 2007).…”

“…Inhibition of GlyRs by a competitive antagonist should be sensitive to changes in the amount of glycine released (Clements, 1996) because SR95531 has an estimated mean lifetime occupancy of 300 s and should unbind within the timescale of synaptic release (Beato et al, 2007). In outside-out or nucleated patches, SR95531 at 70 M, a concentration well below its binding equilibrium constant of 190 M (Beato et al, 2007), blocked the current evoked by 60 M glycine by 61 Ϯ 11% (n ϭ 3), whereas the current evoked by 200 M glycine was reduced only by 10.6 Ϯ 9.5% (data not shown). To mimic fast synaptic release, we recorded currents evoked by brief 1 ms iontophoretic applications of glycine and confirmed that the fraction of the current blocked by 70 M S95531 decreased as the amount of glycine release increased (Fig.…”

“…5B), we examined whether there was a presynaptic change in glycine release by taking advantage of SR95531, a low-affinity competitive antagonist of GlyRs (Wang and Slaughter, 2005;Beato et al, 2007). Inhibition of GlyRs by a competitive antagonist should be sensitive to changes in the amount of glycine released (Clements, 1996) because SR95531 has an estimated mean lifetime occupancy of 300 s and should unbind within the timescale of synaptic release (Beato et al, 2007). In outside-out or nucleated patches, SR95531 at 70 M, a concentration well below its binding equilibrium constant of 190 M (Beato et al, 2007), blocked the current evoked by 60 M glycine by 61 Ϯ 11% (n ϭ 3), whereas the current evoked by 200 M glycine was reduced only by 10.6 Ϯ 9.5% (data not shown).…”