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The intracellular domain of homomeric glycine receptors modulates agonist efficacy.

ABSTRACT: Like other pentameric ligand-gated channels, glycine receptors (GlyRs) contain long intracellular domains (ICDs) between transmembrane helices 3 and 4. Structurally characterized GlyRs are generally engineered to have a very short ICD. We show here that for one such construct, zebrafish GlyR_EM, the agonists glycine, β-alanine, taurine, and GABA have high efficacy and produce maximum single-channel open probabilities greater than 0.9. In contrast, for full-length human α1 GlyR, taurine and GABA were clearly partial agonists, with maximum open probabilities of 0.46 and 0.09, respectively. We found that the elevated open probabilities in GlyR_EM are not due to the limited sequence differences between the human and zebrafish orthologs, but rather to replacement of the native ICD with a short tripeptide ICD. Consistent with this interpretation, shortening the ICD in the human GlyR increased the maximum open probability produced by taurine and GABA to 0.90 and 0.70, respectively, but further engineering it to resemble GlyR_EM (by introducing the zebrafish transmembrane helix 4 and C terminus) had no effect. Furthermore, reinstating the native ICD to GlyR_EM converted taurine and GABA to partial agonists, with maximum open probabilities of 0.66 and 0.40, respectively. Structural comparison of transmembrane helices 3 and 4 in short- and long-ICD GlyR subunits revealed that ICD shortening does not distort the orientation of these helices within each subunit. This suggests that the effects of shortening the ICD stem from removing a modulatory effect of the native ICD on GlyR gating, revealing a new role for the ICD in pentameric ligand-gated channels.

SUBMITTER: Ivica J

PROVIDER: S-EPMC7995613 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Many pentameric ligand-gated ion channels are modulated by extracellular pH. Glycine receptors (GlyRs) share this property, but it is not well understood how they are affected by pH changes. Whole cell experiments on HEK293 cells expressing zebrafish homomeric α1 GlyR confirmed previous reports that acidic pH (6.4) reduces GlyR sensitivity to glycine, whereas alkaline pH (8.4) has small or negligible effects. In addition to that, at pH 6.4 we observed a reduction in the maximum responses to the partial agonists β-alanine and taurine relative to the full agonist glycine. In cell-attached single-channel recording, low pH reduced agonist efficacy, as the maximum open probability decreased from 0.97, 0.91 and 0.66 to 0.93, 0.57 and 0.34 for glycine, β-alanine and taurine, respectively, reflecting a threefold decrease in efficacy equilibrium constants for all three agonists. We also tested the effect of pH 6.4 in conditions that replicate those at the native synapse, recording outside-out currents elicited by fast application of millisecond pulses of agonists on α1 and α1β GlyR, at a range of intracellular chloride concentrations. Acidic pH reduced the area under the curve of the currents, by reducing peak amplitude, slowing activation and speeding deactivation. Our results show that acidification of the extracellular pH by one unit, as may occur in pathological conditions such as ischaemia, impairs GlyR gating and is likely to reduce the effectiveness of glycinergic synaptic inhibition. KEY POINTS: Extracellular pH in the central nervous system (CNS) is known to shift towards acidic values during pathophysiological conditions such as ischaemia and seizures. Acidic extracellular pH is known to affect GABAergic inhibitory synapses, but its effect on signals mediated by glycine receptors (GlyR) is not well characterised. Moderate acidic conditions (pH 6.4) reduce the maximum single channel open probability of recombinant homomeric GlyRs produced by the neurotransmitter glycine or other agonists, such as β-alanine and taurine. When glycine was applied with a piezoelectric stepper to outside out patches, to simulate its fast rise and short duration at the synapse, responses became shorter and smaller at pH 6.4. The effect was also observed with physiologically low intracellular chloride and in mammalian heteromeric GlyRs. This suggests that acidic pH is likely to reduce the strength of inhibitory signalling at glycinergic synapses.

Project description:The functional properties of rat homomeric alpha 1 glycine receptors were investigated using whole-cell and outside-out recording from human embryonic kidney cells transfected with rat alpha1 subunit cDNA. Whole-cell dose-response curves gave EC(50) estimates between 30 and 120 microM and a Hill slope of approximately 3.3. Single channel recordings were obtained by steady-state application of glycine (0.3, 1, or 10 microM) to outside-out patches. Single channel conductances were mostly 60-90 pS, but smaller conductances of approximately 40 pS were also seen (10% of the events) with a relative frequency that did not depend on agonist concentration. The time constants of the apparent open time distributions did not vary with agonist concentration, but short events were more frequent at low glycine concentrations. There was also evidence of a previously missed short-lived open state that was more common at lower glycine concentrations. The time constants for the different components of the burst length distributions were found to have similar values at different concentrations. Nevertheless, the mean burst length increased with increasing glycine. This was because the relative area of each burst-length component was concentration dependent and short bursts were favored at lower glycine concentrations. Durations of adjacent open and shut times were found to be strongly (negatively) correlated. Additionally, long bursts were made up of longer than average openings separated by short gaps, whereas short bursts usually consisted of single isolated short openings. The most plausible explanation for these findings is that long bursts are generated when a higher proportion of the five potential agonist binding sites on the receptor is occupied by glycine. On the basis of the concentration dependence and the intraburst structure we provide a preliminary kinetic scheme for the activation of the homomeric glycine receptor, in which any number of glycine molecules from one to five can open the channel, although not with equal efficiency.

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The intracellular domain of homomeric glycine receptors modulates agonist efficacy.

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