A portable site: a binding element for 17β-estradiol can be placed on any subunit of a nicotinic α4β2 receptor

Abstract

Endogenous steroids can modulate the activity of transmitter-gated channels by directly interacting with the receptor. 17β-Estradiol potentiates activation of neuronal nicotinic α4β2 receptors by interacting with a 4 aa sequence at the extreme C terminus of the α4 subunit, but it is not known whether potentiation requires that the sequence be placed on a specific subunit (e.g., an α4 subunit that is involved in forming an acetylcholine-binding site). By using concatemers of subunits and chimeric subunits, we have found that the C-terminal domain can be moved from the α4 to the β2 subunit and still result in potentiation. In addition, the sequence can be placed on a subunit that contributes to an acetylcholine-binding site or on the structural subunit. The data indicate that this estradiol-binding element is a discrete sequence and suggest that the effect of 17β-estradiol is mediated by actions on single subunits and that the overall consequences for gating occur because of the summation of independent energetic contributions to overall gating of this receptor.

Figure 1.

Schematic views of the receptor and subunits. The top panel shows the overall arrangements in pentamers containing concatemers. Subunits are arranged around the ion channel (dotted circle). ACh-binding sites are indicated by the diamonds between an α4 subunit (contributing the positive or + side of the interface) and a β2 subunit. Four subunits contribute to the agonist-binding sites, whereas the fifth occupies a “structural” position. The concatemers are indicated with linkers connecting the C terminus (denoted by a circle with C) to the N terminus, as shown by the arrowheads. The fainter diamonds on the free subunit assembled with the α/β concatemer indicate that the site is located on opposite sides, depending on the nature of the N-terminal sequence of the free subunit (note that the structural subunit in this combination is contributed from one of the concatemers). The bottom panel shows the constructs used. The top pair shows sequence for the chimeras α-M3-β and β-M3-α. The line above the sequences shows the position of the C-terminal end of the M3 region. The joining point is indicated by the arrow; the sequences were swapped for all positions including and following the indicated residue. The bottom pair shows the βM4→C and βWLAGMI constructs. The line shows the position of the predicted M4 region. The bold letters indicate amino acid residues in the predicted M4 region that differ between α4 and β2.

Figure 2.

The free subunit participates in the assembled receptor, as indicated by responses to agonists. The relative response to 5I A85380 is plotted against the EC₅₀ for ACh; when the receptor contains three β2 subunit N-terminal domains, the relative response is large, and the EC₅₀ is small (free subunits with an α4:β2 ratio of 1:8; black square). Conversely, when two β2 subunits are present, the relative response is small and the EC₅₀ is large (free subunits with an α4:β2 ratio of 8:1; black triangle). The hollow squares show data for combinations in which it is predicted that three β2 subunit N-terminal domains would be present if receptors contained two copies of a concatemer and a single copy of the free subunit, whereas hollow triangles show data for receptors with two predicted β2 N-terminal domains. The black circles show data for the two concatemers expressed without a free subunit. The means of values with two predicted β2 N termini are 5I A85380 relative response of 0.30 ± 0.04 (N = 14 combinations) and EC₅₀ = 97 ± 9 μm, whereas with three predicted β2 N termini the means are 1.47 ± 0.08 (N = 9 combinations) and 6.2 ± 2.7 μm. The means differ at p < 10⁻⁶ for each parameter. Data show mean ± SE, for 27 combinations of constructs (data from 3 or more oocytes), including concatemers alone and combinations of free subunits. The full data set is shown in supplemental Table 1 (available at www.jneurosci.org as supplemental material).

Figure 3.

Concatemers do not appear to be significantly degraded. The panels show two blots of the same transfer of proteins extracted from four batches of oocytes, injected with different constructs on the same day. In A, the transfer was probed with antibody to α4 (H-133; sc5591). In B, the transfer was stripped and reprobed with antibody to β2 (C-20; sc1449). Note that there appears to be some breakdown of α4 in extracts from oocytes injected with α4&β2 subunits. However, it does not appear that concatemers break down to a significant extent. For each preparation, 50 oocytes were used. Approximately 230 μg (∼20% of preparation) of protein loaded in lanes 2, 3, and 4, and ∼50 μg in lane 1 (∼5% of prep). These images are representative of eight gels from three protein preparations. Images are shown in grayscale and reversed intensity scale to allow visualization of minor bands.

Figure 4.

Activation by 5I A85380 indicates positions of subunits in receptors containing concatemers. A shows the relative gating (normalized to the response to 1 mm ACh for the tested cell) for α/β concatemers assembled with wild-type β2 (black triangles and solid black line) or with β2F119Q (hollow triangles and dashed line). The parameters for a Hill equation fit to the data are as follows: EC₅₀, 8 ± 1 nm; maximal response, 1.44 ± 0.07-fold for β2; and EC₅₀, 127 ± 14 nm; maximal response, 1.13 ± 0.11-fold for β2F119Q (the Hill coefficient was constrained to 1 for fits). The hollow circles show responses of the α4/β2 concatemer expressed alone. B shows comparable data for the β/α concatemer expressed with β2 or β2 F119Q. The parameters for a Hill equation fit to the data are as follows: EC₅₀, 29 ± 12 nm; maximal response, 1.44 ± 0.10-fold for β2; and EC₅₀, 16 ± 1 nm; maximal response, 1.29 ± 0.04-fold for β2F119Q (the Hill coefficient was constrained to 1 for fits). Fit parameters and data points are mean ± 1 SE, for data from six cells.

Figure 5.

The responses to ACh and ACh plus 10 μm 17β-estradiol are shown for oocytes injected with selected combinations of subunits. The times of drug application are indicated by the horizontal bars above the traces: the top bar shows the application of a low concentration of ACh alone, whereas the bottom bar shows the application of ACh plus 10 μm 17β-estradiol. The left column shows responses from oocytes injected with the α/β concatemer and different free subunits; in this combination the free subunit contributes to an ACh-binding site. Note that a single copy of wild-type α4 produces a receptor that is potentiated by 17β-estradiol, whereas β2 does not, and swapping the portions of the subunit from the end of M3 to the C terminus transfers potentiation or lack thereof. Calibration (top panel): 10 s (all panels); 260 nA (α/β&α4), 34 nA (α/β&β2), 29 nA (α/β&α-M3-β), 14 nA (α/β&β-M3-α). The right column shows responses from oocytes injected with the β/αWLAAC concatemer and different free subunits; in this combination, the free subunit occupies the structural position. Note that a single copy of wild-type α4 produces a receptor that is potentiated by 17β-estradiol, whereas β2 does not, and swapping the last six residues from α4 to β2 transfers potentiation. Calibration (top panel): 10 s (all panels); 1800 nA (β/αWLAAC&α4), 68 nA (β/αWLAAC&β2), 11 nA (β/αWLAAC&βWLAGMI).

Figure 6.

Transferring the β2 C terminus to α4 removes potentiation by 17β-estradiol, and transferring the α4 C-terminal domain to β2 confers potentiation. Combinations of constructs were chosen in which there are no free WLAGMI domains except in the added free subunit. When expressed with the α/β concatemer, the free subunit occupies a position in which it contributes to an ACh-binding site, whereas when expressed with the β/αWLAAC concatemer it occupies the structural position (Fig. 1). The figure shows the mean response ratio in the presence of 10 μm 17β-estradiol (+1 SE). The labels for the bar show the combination of constructs expressed and the number in parentheses shows the number of free C-terminal domains in the postulated pentamer. The significance levels are shown on the right for the probability that the ratio differs from 1 (no effect, shown by the heavy dashed line): ns, p > 0.05; *p < 0.05; **p < 0.01; ***p < 0.001. The numbers of oocytes tested is shown in parentheses after the significance level. The two dashed lines indicate a response ratio of 1 (no effect; heavy line) or the mean level for free wild-type α4 expressed with that concatemer (thin line). Full data are shown in supplemental Table 2 (available at www.jneurosci.org as supplemental material).

Figure 7.

An increased number of free WLAGMI domains results in increased maximal potentiation by 17β-estradiol. Potentiation is plotted against the concentration of 17β-estradiol for eight combinations of constructs. A shows data for receptors containing one (β/αLAAC&βWLAGMI and α/β&α4; solid lines show fits) or three (α4&β2 8:1 and β/α&βM4→C; dash-dot lines) free WLAGMI domains. B shows data for two (αWLAAC&βWLAGMI 8:1 and β/α&β-M3-α; dash-dot-dot lines) or five (α4&β2WLAGMI 8:1 and α4&βM4→C 8:1; dashed lines) domains. The number of free WLAGMI domains in each combination is shown in parentheses. The lines show fits of the equation r = 1 + R_max([βEst]/(EC₅₀ + [βEst]), where R is the response ratio, R_max is the maximal ratio, [βEst] is the concentration of 17β-estradiol, and EC₅₀ is the concentration producing a half-maximal effect. (The Hill coefficient was constrained to 1.) The fit values are shown in Table 2. The points show the mean ± SE, and the dashed line at a ratio of 1 shows no effect.

Figure 8.

An increased number of free WLAGMI domains results in increased maximal potentiation by 17β-estradiol. The fit R_max + 1 is plotted against the number of free WLAGMI domains predicted to be in the assembled receptor. The data were fit with two simple equations. The first is a linear increase (R_max + 1) = 1 + sM, where R_max is the fit maximal potentiation, s is the constant of proportionality, and M is the number of free WLAGMI domains. The second is a geometric increase (R_max + 1) = r^M, where r is the relative increase in R_max conferred by adding one WLAGMI domain. The lines show the predicted dependence (dashed linear, solid geometric) and the dashed lines show ±1 SE of the fits. The fit values are s = 1.6 ± 0.1 (best fit ± 1 SE of parameter estimate) and r = 1.62 ± 0.02. The geometric fit was better than the linear fit (p = 0.02, F test). Points show mean ± SE for data shown in Table 2. Note that the symbol is sometimes larger than the error bar, and that the two data points at two free WLAGMI overlap. The symbols match those in Figure 7.