Figure 1.
Modulation of different GlyR subtypes by ECs.
(A) Glycine-activated membrane currents through wild-type α1, α2 and α3 GlyRs under control conditions (black) and in the presence of AEA, NA-Gly and VIR (red; all 10 µM). Membrane currents were activated by equipotent (EC10) glycine concentrations for each particular subunit. Chemical structures for the ligands are also shown. (B) Concentration-response curves. (C) Summary of the EC-mediated allosteric modulation of GlyRs subunits obtained at 10 µM concentration. Note that all acidic ECs tested still potentiated the α1 GlyR currents, but inhibited currents through α2 - α3 GlyRs. NOLE, noladin ether; AEA, anandamide; NA-5HT, arachidonyl serotonin; NADA, N-arachidonyl dopamine, NA-Gly; N-arachidonyl glycine; NA-GABA, N-arachidonyl-GABA; NA-Ser, N-arachidonoyl-L-serine; NALA, N-arachidonoyl-L-alanine; AA, arachidonic acid; VIR, virodhamine. Data are means ± SEM from 6-15 cells.
Figure 2.
NA-Gly effects on chimeric GlyR constructs.
(A) Schematic depiction of wild type and chimeric GlyRs. (B) Examples of whole-cell currents recorded from α1α2 or α2α1 GlyRs before (black) and during the application of NA-Gly (10 µM, red). (C) Percent change of the normalized glycinergic membrane currents during the application of NA-Gly (10 µM) using equipotent (EC10) glycine concentrations. The exchange of the IL between TM3 and TM4 plus the TM4 domain between α1 and α2 GlyRs did not significantly influence the NA-Gly-induced modulation.
Figure 3.
Positive and negative NA-Gly allosteric effects on α1 and α2 GlyRs are influenced by a single TM2 domain residue.
(A) Primary sequence alignment between α1 and α2 GlyR subunits from TM1 to TM3 regions. The 3 non-conserved residues are highlighted by green boxes. (B) Examples of current traces through GlyRs with mutated TM domains, in absence (black) or presence of NA-Gly (red) (C) The bar graphs summarizes the normalized glycine-evoked current enhancement after the application of 10 µM NA-Gly on α1 GlyRs with mutations in specific residues within the TM domains (D) Schematic representation of wild-type and TM2-mutated GlyRs (E) Concentration-response curves for NA-Gly in wild-type and TM-mutated α1 and α2 GlyRs using two different agonist concentrations. Note that the specific mutation G254A in α1 GlyRs significantly attenuated the EC potentiation, whereas the reverse TM2 mutation in α2 GlyRs (A261G) additionally decreased the NA-Gly-induced inhibition.
Table 1.
Electrophysiological properties of wild-type and mutated GlyRs.
Figure 4.
The composition of extracellular loop 2 influences the NA-Gly-induced potentiation of α1 GlyRs.
(A) Amino acid sequence alignment between α1 and α2 GlyRs within the extracellular loop 2. The A52 residue in α1 GlyRs and their homologous position in α2 GlyRs are highlighted by a green box. (B) Schematic depictions of GlyRs with point mutations in the extracellular loop 2 (C) Concentration-response curves for NA-Gly in wild-type and extracellular loop 2-mutated α1 and α2 GlyRs using two different agonist concentrations. The mutation A52T significantly attenuated the NA-Gly sensitivity of α1 GlyRs, whereas the reverse mutation in α2 GlyRs (T59A) did not alter NAGly-induced inhibition.
Figure 5.
Selective extracellular loop 2 and TM domain mutations in α2 GlyRs convert NA-Gly into an allosteric potentiator.
(A) Examples of glycine-activated current traces from wild-type α1 and mutant α2 T59A/A261G/A303S GlyRs in the presence of NA-Gly (in red) (B) Summary of the effects of NA-Gly after simultaneous extracellular loop 2 and TM reverse mutations on α2 GlyRs (*** P<0.001, vs α2 GlyRs) (C) Schematic diagrams of the triple mutated α1 and α2 GlyRs (D) Sensitivity of the normalized glycine-activated currents elicited in wild-type and triple mutated GlyRs to different concentrations of NA-Gly. Note that three simultaneous reverse mutations in α2 GlyR converted NA-Gly into an allosteric potentiator, whereas the homologous substitutions within α1 GlyR still did not produce a significant inhibition.
Figure 6.
The negative allosteric effects of NA-Gly on α3 GlyRs were attenuated but not reverted by altering the TM2 domain composition.
(A) Primary sequence alignment of α1, α2 and α3 GlyR subunits in selected extracellular loop 2, TM2 and TM3 segments (B) Schematic depiction of wild type and point mutated α3 GlyRs. (C) Examples of current traces through wild-type and point-mutated α3 GlyRs in absence (black) or presence of NA-Gly (10 µM, red) (D) Concentration-response curves for NA-Gly in wild-type and TM2-mutated α3 GlyRs using two different glycine concentrations. Note that this specific mutation A265G in α3 GlyRs significantly attenuated the NA-Gly-induced inhibition, but did not convert the inhibition into potentiation
Figure 7.
The positive allosteric modulation elicited by NA-Gly is influenced by a conserved lysine residue within the α1 GlyR large intracellular loop.
(A) The schematic receptor representation and the primary sequence alignment describe the position of the conserved intracellular K385 residue within the GlyR structure (B) Glycine-activated current traces from wild-type or K385A-mutated α1 GlyRs before (black) and during the application of NA-Gly (5 µM, red) (C) Concentration-response curves for NA-Gly obtained from wild-type and K385-mutated GlyRs. The intracellular mutation significantly attenuated the NA-Gly-induced potentiation of α1 GlyRs.
Figure 8.
A conserved intracellular lysine residue determines the positive allosteric modulation by neutral ECs on GlyRs.
(A) Examples of current traces through wild-type α1 GlyRs and K385A mutated GlyRs in absence (black) or presence of AEA (5 µM, red) (B) Sensitivity to AEA of the normalized glycine-activated currents in wild-type and K385A-mutated GlyRs. The intracellular mutation effectively attenuated the AEA-induced modulation of the three GlyR subunits. (C) Glycine-activated current traces from wild-type or K385A-mutated α1 GlyRs before (black) and during the application of NA-5HT (5 µM, red) (D) Concentration-response curves for NA-5HT obtained from wild-type and K385-mutated α1 GlyRs (E) Summary of the allosteric effects elicited by AEA and NA-5HT in wild-type and K385A-mutated GlyRs. The current potentiation was significantly attenuated by the intracellular mutation. ***, P<0.001 between each wild-type GlyR and its corresponding K385A mutant.
Figure 9.
Molecular sites for the allosteric modulation of different GlyR subtypes by ECs.
The schematic diagram summarizes the molecular sites for the allosteric modulation of GlyRs by acidic and neutral ECs. The inhibition of α2 and α3 GlyRs elicited by NA-Gly was specifically influenced by a single TM2 residue (A261 in α2 GlyRs and A265 in α3 GlyRs), whereas the NA-Gly-induced potentiation of α1 GlyRs was reduced by mutating loop 2 (A52), TM2 (G254) or intracellular (K385) amino acids. On the other hand, the AEA-induced potentiation of these three GlyR subtypes was reduced by mutating a conserved intracellular lysine residue (K385 in α1 GlyRs).