Activation of human alpha1 and alpha2 homomeric glycine receptors by taurine and GABA

Abstract

1. Two ligand binding alpha subunits, alpha1 and alpha2, of the human (H) glycine receptor (GlyR) are involved at inhibitory synapses in the adult and neonatal spinal cord, respectively. The ability of homomeric alphaH1 and alphaH2 GlyRs to be activated by glycine, taurine and GABA was studied in Xenopus oocytes or in the human embryonic kidney HEK-293 cell line. 2. In outside-out patches from HEK cells, glycine, taurine and GABA activated both GlyRs with the same main unitary conductance, i.e. 85 +/- 3 pS (n = 6) for alphaH1, and 95 +/- 5 pS (n = 4) for alphaH2. 3. The sensitivity of both alphaH1 and alphaH2 GlyRs to glycine was highly variable. In Xenopus oocytes the EC50 for glycine (EC50gly) was between 25 and 280 microM for alphaH1 (n = 44) and between 46 and 541 microM for alphaH2 (n = 52). For both receptors, the highest EC50gly values were found on cells with low maximal glycine responses. 4. The actions of taurine and GABA were dependent on the EC50gly: (i) their EC50 values were linearly correlated to EC50gly, with EC50tau approximately 10 EC50gly and EC50GABA approximately 500-800 EC50gly; (ii) they could act either as full or weak agonists depending on the EC50gly. 5. The Hill coefficient (n(H)) of glycine remained stable regardless of the EC50gly whereas n(H) for taurine decreased with increasing EC50tau. 6. The degree of desensitization, evaluated by fast application of saturating concentrations of agonist on outside-out patches from Xenopus oocytes, was similar for glycine and taurine on both GlyRs and did not exceed 50 %. 7. Our data concerning the variations of EC50gly and the subsequent behaviour of taurine and GABA could be qualitatively described by the simple del Castillo-Katz scheme, assuming that the agonist gating constant varies whereas the binding constants are stable. However, the stability of the Hill coefficient for glycine was not explained by this model, suggesting that other mechanisms are involved in the modulation of EC50.

Figure 1. Variability of the EC₅₀ for glycine of human homomeric αH1 and αH2 GlyRs

A, left: glycine-induced whole-cell currents from the most (upper traces) and the least (lower traces) sensitive oocytes expressing αH1 GlyRs recorded in this study. Concentrations are indicated above each trace. A, right: concentration dependencies for the same cells fitted by the Hill equation. The lowest EC_50gly of αH1 was 25 μm, the highest 280 μm. B, similar presentation as in A for data recorded from oocytes expressing αH2 GlyRs. The lowest and highest EC_50gly were 46 and 541 μm, respectively.

Figure 2. Dependence of EC_50gly and I_maxgly on the amount of cDNA injected into oocytes

A, distribution of the EC_50gly values (ordinates) for different amounts of injected cDNA (27, 40, 50 and 270 pg; abscissa). ⋄, mean EC_50gly for each amount of cDNA. Note the nearly 10-fold variation in EC_50gly for oocytes injected with 270 pg cDNA. B, distribution of the ratio I_maxgly/V_h (with I_maxgly in nA (200-10 000 nA) and with V_h = -60 or -70 mV) for various amounts of injected cDNA. ⋄, mean ratios.

Figure 3. Relationship between the EC_50gly for glycine and the maximum glycine response

The EC_50gly of each oocyte expressing either αH1 GlyR (○) or αH2 GlyR (•) was plotted against the maximum glycine response relative to the holding potential (i.e. I_maxgly/V_h expressed in nS).

Figure 4. Single-channel currents evoked by glycine, taurine and GABA in outside-out patches excised from HEK 293 cells

A, left: patch containing αH1 GlyRs. Currents induced by the three agonists at the concentrations indicated above each trace recorded at V_h = -70 mV. Note that currents obtained with glycine, taurine and GABA had similar amplitudes. A, right: I-V relationship of single-channel currents induced on the same patch by the three agonists. Only the main conductance for each is presented. At hyperpolarizing potentials, the conductance slope was 86 pS. B, same presentation as in A but for αH2 GlyRs. Typical traces (V_h = -60 mV) of agonist-induced currents are shown on the left. The I-V relationships from this patch (right) indicate that the three agonists activated a similar main conductance of 97 pS.

Figure 5. Glycine, taurine and GABA responses of highly sensitive αH1 and αH2 GlyRs

A and B, maximal currents induced by saturating concentrations of glycine, taurine and GABA and dose-response curves from two Xenopus oocytes expressing typical highly sensitive αH1 (A) and αH2 (B) subunits. EC₅₀ values of the three tested agonists are indicated. The relative maximum responses of taurine and GABA, determined as I_maxagonist/I_maxgly from the illustrated traces, were 90 % and 69 % for the αH1 GlyR and 86 % and 73 % for the αH2 GlyR, respectively. Holding potentials were -70 mV.

Figure 6. Glycine, taurine and GABA responses of poorly sensitive oocytes expressing either αH1 or αH2 subunits

A and B, presentation as in Fig. 5 of two oocytes expressing poorly sensitive αH1 (A) or αH2 (B) GlyRs. EC₅₀ values are indicated above each dose-response curve, except for GABA (not determined). V_h = -60 mV. I_maxtau/I_maxgly = 9 % for αH1 and 34 % for αH2. I_maxGABA/I_maxgly = 4 % and 6 %, for αH1 and αH2, respectively.

Figure 7. Relationship between the EC₅₀ for glycine and for taurine and GABA of the αH1 and αH2 GlyRs

A and B, linear relationships between the EC₅₀ values for glycine and taurine (A) or GABA (B) for both αH1 (○) and αH2 (•) GlyRs. The data were approximated by the following equations: EC_50tau =a+ 10.3EC_50gly, R = 0.94 (for αH1) and EC_50tau =a+ 9.8EC_50gly, R = 0.92 (for αH2); EC_50GABA =a+ 483EC_50gly, R = 0.95 (for αH1) and EC_50GABA =a+ 765EC_50gly, R = 0.64 (for αH2). R, coefficient of correlation. C, relationships between the EC₅₀ for glycine (abscissa) and for taurine and GABA (ordinate) for both αH1 (○) and αH2 (•). For convenience of illustration, data are plotted on logarithmic scales.

Figure 8. Relative maximum responses of taurine and GABA depend on the EC_50gly of the GlyRs

The relative maximum responses of taurine (determined as I_maxtau/I_maxgly; A) and of GABA (I_maxGABA/I_maxgly; B) from oocytes expressing αH1 or αH2 GlyRs were plotted against the EC_50gly. Note the similarities between the two GlyRs.

Figure 9. Relationships between the EC₅₀ for glycine and taurine and their Hill coefficient

The Hill coefficient (n_H) of glycine (A) and taurine (B) were plotted against the EC₅₀ of these agonists for both αH1 and αH2 GlyRs. Note that only the n_H for taurine decreased as the EC₅₀ increased.

Figure 10. Estimation of the desensitization from outside-out currents activated by glycine, taurine and GABA

A and B, currents induced by high concentrations of glycine (1 mm), taurine (10 or 30 mm) and GABA (50 or 100 mm) applied on outside-out patches from Xenopus oocytes using a fast perfusion system. V_h = -20 mV. C, responses of taurine and GABA normalized to the I_gly. For each patch, the ratio I_agonist/I_gly was measured either at the peak (□) or at the end of the pulse (I_{500 ms}, ▪). Results are given ±s.d. Asterisk indicates a statistical difference between the mean ratio at the peak and at 500 ms (P < 0.001, t test). D, degree of desensitization of the currents induced by the three agonists, given by the ratio of the amplitude at 500 ms (I_{500 ms}) and the current at the peak (I_peak). Results are given ±s.d. Only patches on which all three agonists could be tested were included in the analysis.

Figure 11. Comparison of the relative maximum responses of taurine on wild-type αH1 GlyRs and on mutant αH1 GlyRs

The ratio I_maxtau/I_maxgly obtained in this study on αH1 GlyRs (•) are compared with those observed by Lynch et al. (1997, ○) on mutant and wild-type αH1 GlyRs expressed in transfected HEK cells and with those from Schmieden et al. (1992, ; □) on wild-type and mutated αH1 GlyRs expressed in Xenopus oocytes. Data from mutants I244A and W243A which exhibit an accelerated desensitization (Lynch et al. 1997) were not included in the graph.

Figure 12. Predictions from the del Castillo-Katz scheme when the binding constant (K_A) remains stable while the gating constant (E) varies

A, dependence of P_maxgly on c_50gly. c₅₀ is defined as the EC₅₀ normalized to its binding constant and P_max is the maximum fraction of open channel. Note the similar shape of this curve and the experimental results presented in Fig. 3B., distribution of the c₅₀ for taurine and GABA as a function of the c₅₀ for glycine. Considering our result (Fig. 7), the efficacies of taurine and GABA were fixed here and in C to E_tau = 0.1 ×E_gly and E_GABA = 0.002 ×E_gly. Note the similarity with Fig. 7C. C., maximum fraction of open channel by taurine or by GABA relative to P_maxgly decline with increasing c_50gly. Note the similarities with Fig. 8.