An N-terminal histidine regulates Zn(2+) inhibition on the murine GABA(A) receptor beta3 subunit

Abstract

1. Whole-cell currents were recorded from Xenopus laevis oocytes and human embryonic kidney cells expressing GABA(A) receptor beta3 subunit homomers to search for additional residues affecting Zn(2+) inhibition. These residues would complement the previously identified histidine (H267), present just within the external portal of the ion channel, which modulates Zn(2+) inhibition. 2. Zinc inhibited the pentobarbitone-gated current on beta3(H267A) homomers at pH 7.4, but this effect was abolished at pH 5.4. The Zn(2+)-sensitive spontaneous beta3 subunit-mediated conductance was also insensitive to block by Zn(2+) at pH 5.4. 3. Changing external pH enabled the titration of the Zn(2+) sensitive binding site or signal transduction domain. The pK(a) was estimated at 6.8 +/- 0.03 implying the involvement of histidine residues. 4. External histidine residues in the beta3 receptor subunit were substituted with alanine, in addition to the background mutation, H267A, to assess their sensitivity to Zn(2+) inhibition. The Zn(2+) IC(50) was unaffected by either the H119A or H191A mutations. 5. The remaining histidine, H107, the only other candidate likely to participate in Zn(2+) inhibition, was substituted with various residues. Most mutants were expressed at the cell surface but they disrupted functional expression of beta3 homomers. However, H107G was functional and demonstrated a marked reduction in sensitivity to Zn(2+). 6. GABA(A) receptor beta3 subunits form functional ion channels that can be inhibited by Zn(2+). Two histidine residues are largely responsible for this effect, H267 in the pore lining region and H107 residing in the extracellular N-terminal domain.

Figure 1

Inhibition of spontaneous Cl⁻ currents through β3 subunit GABA_A receptors by Zn²⁺ and sequence alignments of external histidine residues for GABA_A and GABA_C receptor subunits. (A) Membrane currents recorded from wild-type β3 and mutant β3(H267A) subunits expressed in oocytes held at −40 mV. Membrane conductances in the presence and absence of Zn²⁺ were assessed using hyperpolarizing voltage commands (−10 mV, 1 s, 0.2 Hz). (B) Zinc concentration inhibition relationships for blocking the spontaneous Cl⁻ current transduced by β3 wild-type and β3^H267A mutant GABA_A receptors. The IC₅₀s were 0.15±0.01 μM (β3) and 199±13 μM (β3^H267A). Data points represent the mean±s.e. from seven cells. (C) Schematic diagram of the GABA_A receptor β3 subunit illustrating the transmembrane domains (TM) and external N- and C-termini and large intracellular loop between TM3 and TM4. The external histidine residues and single histidine in TM2 are highlighted. (D) sequence alignments of part of the N-terminal extracellular domain and TM1–TM3 for GABA_A receptor α1, α6, β1 and β3 subunits, and the GABA_C receptor subunit, ρ1. Histidines are depicted as bold characters and those previously demonstrated to affect Zn²⁺ modulation are also italicized.

Figure 2

Sensitivity of Zn²⁺ regulation of β3^H267A GABA_A receptor subunits expressed in oocytes to external pH. Concentration response curves were constructed for pentobarbitone (PB) modulated currents and normalized to the response evoked by 50 μM PB, in the absence and presence of 300 μM Zn²⁺ recorded from oocytes expressing β3^H267A homomers in Ringer at pH 7.4 (A) and 5.4 (B). Curves were fit to the data as described in the methods (n=3). (C) Zinc concentration inhibition relationship for spontaneous currents mediated by β3^H267A homomers in oocytes exposed to Ringer at pH 7.4 and 5.4. Data were obtained from n=5 cells.

Figure 3

Modification of histidine residues in β3 subunits by diethylpyrocarbonate affects Zn²⁺ inhibition. (A) Membrane currents modulated by PB and Zn²⁺ in HEK cells expressing β3^H267A mutants in the absence and presence of diethylpyrocarbonate (DEPC). Drugs were applied for the duration indicated by the lines. (B) Bargraph of PB-modulated current for β3^H267A mutants following exposure to 1 mM PB; +100 μM Zn²⁺; after recovery; following continuous application of 1 mM DEPC with either PB or PB+Zn²⁺ (n=3).

Figure 4

Effect of mutating histidine residues H119 and H191 in GABA_A receptor β3^H267A homomers on Zn²⁺ inhibition. Concentration response curves for PB were established for β3, β3^H267A and either β3^{H119A, H267A} (A) or β3^{H191A, H267A} (C) GABA_A receptor constructs expressed in oocytes. Data were normalised to the maximum response for each construct and obtained from n=11 cells. Concentration inhibition relationships for Zn²⁺ antagonizing the spontaneous Cl⁻ current was determined for β3, β3^H267A and either β3^{H119A, H267A} (B) or β3^{H191A, H267A} (D) constructs. Data are mean±s.e.mean from n=8 cells.

Figure 5

Zn²⁺ sensitivity of the β3^{H119A,H191A,H267A} GABA_A receptor is affected by external pH. In expressing oocytes, control responses to 100 μM PB were recorded in the presence of 200 μM Zn²⁺ over the external Ringer pH range 5.4 to 8.4 and presented as a pH titration. The reduced inhibitory effect of Zn²⁺ as the external pH increased was determined as a percentage of the control PB response at each pH in the absence of Zn²⁺. The curve was generated according to the inhibition model described in the methods. The pK_a determined from the curve fit was 6.8±0.1 (n=3).

Figure 6

Confocal microscopy of the cell surface expression of H107X mutants. The panels illustrate HEK cells expressing wild-type β3 homomers (A) and β3 receptor mutants: H107A (B), H107G (C), H107K (D) after exposure to bd17 antisera. Staining for the β3(H107X) mutants revealed their relative location between cell surface membrane and intracellular compartments. Scale bar represents 10 μm.

Figure 7

Ablation of inhibition by Zn²⁺ on H107G β3 subunit mutants. (A) Membrane currents recorded from β3^{H107G, H267A} expressing HEK cells modulated by 1 mM PB in the absence and presence of 1 mM Zn²⁺. (B) Zn²⁺ inhibition concentration response curves for β3 wild-type, β3^H267A and β3^{H107G, H267A} subunit receptors. All data were fitted according to the methods (n=5).