In glycine and GABA(A) channels, different subunits contribute asymmetrically to channel conductance via residues in the extracellular domain

Abstract

Single-channel conductance in Cys-loop channels is controlled by the nature of the amino acids in the narrowest parts of the ion conduction pathway, namely the second transmembrane domain (M2) and the intracellular helix. In cationic channels, such as Torpedo ACh nicotinic receptors, conductance is increased by negatively charged residues exposed to the extracellular vestibule. We now show that positively charged residues at the same loop 5 position boost also the conductance of anionic Cys-loop channels, such as glycine (α1 and α1β) and GABA(A) (α1β2γ2) receptors. Charge reversal mutations here produce a greater decrease on outward conductance, but their effect strongly depends on which subunit carries the mutation. In the glycine α1β receptor, replacing Lys with Glu in α1 reduces single-channel conductance by 41%, but has no effect in the β subunit. By expressing concatameric receptors with constrained stoichiometry, we show that this asymmetry is not explained by the subunit copy number. A similar pattern is observed in the α1β2γ2 GABA(A) receptor, where only mutations in α1 or β2 decreased conductance (to different extents). In both glycine and GABA receptors, the effect of mutations in different subunits does not sum linearly: mutations that had no detectable effect in isolation did enhance the effect of mutations carried by other subunits. As in the nicotinic receptor, charged residues in the extracellular vestibule of anionic Cys-loop channels influence elementary conductance. The size of this effect strongly depends on the direction of the ion flow and, unexpectedly, on the nature of the subunit that carries the residue.

FIGURE 1.

Effects on the single channel slope conductance of α1 homomeric GlyR of a charge reversal mutation of the extracellular domain conductance determinant in loop 5. A and C are examples of inward (A) and outward (C) currents (low-pass filtered at 1 kHz) recorded from HEK cells in the cell-attached configuration in the presence of 0.2 mm Gly. Imposed holding potentials are as indicated. For inward currents only (recorded in a Na⁺-based external solution) the true transmembrane potential will depend also on the cell resting membrane potential. Outward currents were recorded in K⁺-based external solution, which effectively abolishes the cell resting potential. The baseline is indicated by a dashed line for each trace. B and D are current-voltage relationships for wild type (continuous line, Δ) and mutant receptors (dashed line, ●), for inward and outward currents, respectively. E, alignment of loop 5 of Torpedo α1, GABA_A, and Gly subunits. The conserved charged residues are bold and highlighted in gray. Charge reversal mutations at a position homologous to Asp-97 of the α1 Torpedo (6) replace Lys with Glu and are indicated throughout by the superscript KE.

FIGURE 2.

Effects on the single channel slope conductance of the α1β heteromeric GlyR of charge reversal mutations in the loop 5 extracellular conductance determinant. Representative recordings of inward (A) and outward (C) currents recorded from HEK cells in cell-attached mode at different voltages (display f_c = 1 kHz). B and D are inward and outward current-voltage relationships for wild type and mutant receptors: α1β (Δ), α1^KEβ (●), α1β^KE (▿), α1^KEβ^KE (□). Note that inward currents are insensitive to charge reversal mutations in this position, even when all five subunits carry the mutation. On the contrary, the conductance of outward currents is markedly decreased by this mutation, but only when the mutation is carried either by the α1 or by both the α1 and the β subunits. Mutating the β subunit on its own does not affect outward slope conductance. Slope conductance values for each subunit combination are reported in Table 2.

FIGURE 3.

Effects of charge reversal mutations on the outward conductance of α1β heteromeric Gly receptors expressed by coinjection of the α1 and β subunit constructs, or constrained to contain two α and three β subunits by means of concatenated constructs. (α1_β) tandem subunit constructs were co-injected with a monomeric β subunit into Xenopus oocytes and the outward currents were recorded at different voltages. A shows examples of traces recorded at positive voltages for different combinations of wild-type and mutated subunits (display f_c = 1 kHz). Slope conductance values for each combination of mutant and wild-type subunits are reported in Table 3. B shows current-voltage plots for wild type and mutant receptors. α1β (Δ), α1^KEβ (●), α1β^KE (▿),α1^KEβ^KE (□). Mutations carried by the β subunit had no effect on conductance, even though the stoichiometry of this receptor is 2α:3β. C shows examples of traces for the same wild type and mutant combinations expressed by co-injecting un-tethered α1 and β subunits. D shows current-voltage plots for wild-type and mutant un-tethered receptors.

FIGURE 4.

In the α1β Gly channels the pattern of effect of mutations in the different subunits is not altered by increasing permeant ion concentration. Representative traces of outward currents recorded in cell-attached patches at different voltages (display f_c = 1 kHz) in 300 mm chloride (solution S4 in Table 1). A shows example of traces recorded at positive voltages for different combinations of wild-type and mutated subunits for the heteromeric α1β Gly channels. B, outward current-voltage relationships for the wild-type and mutant heteromeric α1β receptor: α1β (Δ), α1β^KE (▿), α1^KEβ (●), α1^KEβ^KE (□). C, summary of outward slope conductance values for GlyRs with 3α:2β (HEK, upper panel) and 2α:3β (expressed as concatenated subunits in oocytes, lower panel) stoichiometries. Values are normalized to the conductance value of the wild type. Note that when both subunits are mutated the conductance decreases more markedly in the 3α form (32.2% of the wild type) rather than in the 2α form of the receptor (45.6% of the wild type).

FIGURE 5.

Charge reversal mutations in the extracellular domain affect outward conductance of the α1β2γ2 GABA_A receptor. α1, β2, and γ2 subunits were expressed in HEK cells at a 1:1:4 ratio. A shows representative outward currents recorded in the cell-attached configuration at several voltages from wild-type and mutant receptors (display f_c = 1 kHz). Each mutant subunit carries two charge reversal mutations (KK to DD) at homologous position to Asp-97 of the α1 Torpedo (see text). Conductance values are reported in Table 5. B, current-voltage plots for representative mutant and wild-type receptors. α1β2γ2 (black line, ●), α1^KDβ2γ2 (red line, x), α1 β2γ2^KD (green line, Δ), α1^KDβ2γ2^KD (violet line and ♢). C, conductance values for all subunit combination shown as bar graph. Introduction of charge reversal mutations at homologous positions in the α1, β2, or γ2 subunit decreases conductance in a subunit-specific manner. Asterisks denote the significance after a one-way ANOVA test followed by Dunnett's test. D, bar graph of outward conductance values normalized for their wild type. The γ2 subunit does not affect conductance when it is the only subunit carrying the charge reversal mutation. However, note the γ2 mutation decreases synergistically conductance in combination with mutations in either the α1 or the β2 subunit.