Inhibition of ELIC by calcium.

<p>(A) ELIC single channel currents in the presence of different extracellular concentrations of Ca²⁺ and all-points amplitude histograms (recordings were from oocyte outside-out patches at −80 mV holding potential). (B) Maximum ELIC responses to saturating concentrations of the agonist cysteamine in the absence and presence of Ca²⁺. Currents were measured from oocytes held at −40 mV under two-electrode voltage clamp. Agonist application is indicated by a bar. Responses to cysteamine in the absence of extracellular Ca²⁺ are followed by responses in the presence of Ca²⁺ (concentration in mM as shown) and by recovery application of cysteamine in the absence of Ca²⁺. (C) Plot of maximum agonist responses and single channel currents at different Ca²⁺ concentrations. The currents are normalized to the control values (in the absence of Ca²⁺). Maximum cysteamine currents (blue symbols) were measured with the two-electrode voltage clamp technique in oocytes as in panel B. Single channel currents (green symbols) were measured in the outside-out configuration as in panel A. (D) Cysteamine dose–response relationships for ELIC in the presence of different concentrations of Ca²⁺. (E) Equilibrium cysteamine binding isotherms determined by ITC for ELIC in the absence (left) and presence of Ca²⁺. Solid curves represent fits to a single-site binding isotherm with Keff = 0.7 mM (no Ca²⁺) and 1.5 mM (0.6 mM Ca²⁺), respectively. (F) Cysteamine dose–response relationships for ELIC in the presence of different concentrations of ACh. (G) Schild plot quantifying the inhibition by Ca²⁺ and ACh. EC₅₀ values were obtained from fits to data shown in panels (D) and (F). Potencies of the antagonists (pA values) were obtained by linear regression, and the intersection with the <i>x</i>-axis is indicated (•). (H) Fraction of the maximum current response at different Ca²⁺ concentrations. The solid line shows a fit to a Langmuir equation with a K_i of 6 mM. The data presented in panels (C), (D), (F), and (H) are averages from at least five oocytes; errors are SD. The solid lines in (D) and (F) show fits to a Hill equation. Currents were recorded at −40 mV. (I) Activation kinetics of macroscopic currents of ELIC activated by propylamine in response to fast solution exchange at different Ca²⁺ concentrations. ELIC was expressed in HEK 293 cells, and currents were recorded from excised patches in the outside-out configuration at −100 mV.</p

Data collection and refinement statistics.

<p>Values in parentheses are for highest resolution shell. R.m.s., root mean square.</p

Activation and deactivation kinetics of ELIC.

<p>Activation and deactivation kinetics of ELIC.</p

Divalent ion inhibition in mutants of the regulatory site S_out.

<p>Dose–response relationships of the ELIC mutants D113A (A) and D158A (B), E150A (C), and the double mutant D113A/D158A (D) activated by cysteamine at different concentrations of Ca²⁺ are shown. (E) Schild plots quantifying the inhibition of ELIC mutants by Ca²⁺. EC₅₀ values were obtained from data shown in panels (A–D). Potencies of the antagonists (pA values) were obtained by linear regression; the intersection with the <i>x</i>-axis is indicated (•). WT is shown for comparison. (F) Graphical depiction of potencies for Ca²⁺ inhibition in different mutants. (G) Maximum current response of the double mutant D113A/D158A at different Ca²⁺ concentrations. The currents are normalized to the maximum response in the absence of Ca²⁺. Whole cell currents measured at −40 mV with the two-electrode voltage clamp technique are shown in red (the averages of at least 5 oocytes are shown; errors are SD). Single channel currents from the double mutant D113A/D158A were measured in the outside-out configuration and are shown in green. WT macroscopic and single channel currents are shown as dashed lines for comparison. (H) Anomalous difference electron density (calculated at 5 Å and contoured at 5 σ) from data of the double mutant D113A/D158A in complex with Ba²⁺ is superimposed on a model of ELIC in ribbon representation. Ion-binding sites are indicated. (I) Dose–response relationships of the double mutant D113A/D158A activated by propylamine at different concentrations of Zn²⁺. The data presented in panels (A–D) and (I) are averages from at least 5 oocytes; errors are SD. The solid lines show fits to a Hill equation. Currents were recorded at −40 mV. A dose–response curve of WT in the absence of Ca²⁺ (dashed line) is shown for comparison.</p

Inhibition of ELIC by different divalent cations.

<p>Dose–response relationships of ELIC activated by cysteamine at different concentrations of Ba²⁺ (A), Sr²⁺ (B), and Mg²⁺ (C) and ELIC activated by propylamine at different concentrations of Zn²⁺ (D). (E) Schild plot quantifying the inhibition by different divalent cations. EC₅₀ values were obtained from fits to data shown in panels (A–D). Potencies of the Antagonists (pA values) were obtained by linear regression, and the intersection with the x-axis is indicated. The data presented in panels (A–D) are averages from at least 5 oocytes; errors are SD. The solid lines show fits to a Hill equation. Currents were recorded at −40 mV.</p

Structure of ELIC in complex with divalent cations.

<p>(A) Sequence of ELIC with secondary structure elements indicated below. Residues contributing to ion coordination in different sites are highlighted (S_in, yellow; S_out, red; S_pore, cyan; Arg 91 in the ligand-binding pocket, S_lig, grey). (B) Anomalous difference electron density of ELIC in complex with Ba²⁺ superimposed on the structure of the ELIC pentamer (shown as ribbon representation). Ion-binding sites are labeled. Close-up of S_pore (C) and S_in (D). The protein is shown as Cα-trace with selected side-chains close to Ba²⁺ (red sphere) shown as sticks. (E) Close-up of S_out. The protein is shown as Cα-trace with selected side-chains close to Ba²⁺ (red sphere) shown as sticks. The 2F_o-F_c electron density of a dataset from a crystal of space group P2₁ was calculated at 3.8 Å and contoured at 1 σ (shown in cyan). The refined model used to calculate phases did not contain Ba²⁺-ions. The anomalous difference electron densities shown in (B–E) (red mesh) were calculated from the same dataset at 5 Å and contoured at 5 σ. Crystals of space group P4₃ showed a qualitatively similar picture. Structures in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001429#pbio-1001429-g003" target="_blank">Figures 3</a>–<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001429#pbio-1001429-g007" target="_blank">7</a> were prepared with DINO (<a href="http://www.dino3d.org" target="_blank">www.dino3d.org</a>).</p

Potential mechanisms.

<p>(A) Interactions in the regulatory divalent ion-binding site of ELIC (left) in comparison with a regulatory Ca²⁺ binding site of the BK-channel (middle) and the Zn²⁺-transporter YiiP (right). (B) Schematic model of a potential mechanism for the inhibitory effect of divalent ions. The two rows show simplified schemes for channel activation in control conditions (top) and in the presence of divalent ions. From left to right, the schemes show that binding of agonist molecules (red ovals) to the extracellular domain (with microscopic affinity <i>K</i>_d) is followed by conformational changes (yellow background) that result in channel opening. Channel gating (described by the efficacy equilibrium constant <i>E</i>) is impaired when the channel is bound to divalent ions (yellow circles, <i>E</i>_Ca²⁺). The decrease in agonist efficacy is likely to be due to a change in the rate of opening, as shown by the size of the arrows in the last step of the reaction. (C) Schematic mechanism of how binding sites located on similar places of an oligomeric channel could alternately stabilize the closed or open conformation of the channel.</p

Divalent ion inhibition in mutants of the nonregulatory sites S_pore and S_in.

<p>Dose–response relationships of the ELIC point mutant R91A (A) activated by cysteamine at different concentrations of Ca²⁺. (B) Anomalous difference electron density (calculated at 5 Å and contoured at 5 σ) from data of the mutant N251A in complex with Ba²⁺ superimposed on a model of ELIC in ribbon representation. Ion-binding sites are indicated. Dose–response relationships of the ELIC point mutants N251A (C), S84A (D), and D86A (E) activated by cysteamine at different concentrations of Ca²⁺. The data presented in panels (A), (B), (D), and (E) are averages from at least 5 oocytes; errors are SD. The solid lines show fits to a Hill equation. Currents were recorded at −40 mV. A dose–response curve of WT in the absence of Ca²⁺ (dashed line) is shown for comparison. (F) Schild plots quantifying the inhibition of ELIC mutants by Ca²⁺. EC₅₀ values were obtained from fits to data shown in panels (A), (B), (D), and (E). Potencies of the antagonists (pA values) were obtained by linear regression; the intersection with the <i>x</i>-axis is indicated (•). WT is shown for comparison.</p

Inhibition by ACh.

<p>(A) Anomalous difference electron density (calculated at 5 Å and contoured at 6 σ) from data of WT in complex with TMAs is superimposed on a model of ELIC in ribbon representation. The ligand-binding site is indicated. Dose–response relationships upon activation with cysteamine of the ELIC mutants R91A (B) and the double mutant D113A/D158A (C) at different concentrations of ACh. (F) Schild plots quantifying the inhibition of ELIC mutants by ACh. EC₅₀ values were obtained from data shown in panels (B–C). Dose–response relationships upon activation with cysteamine of WT in the presence of either 0.25 (E), 0.5 (F), or 1 mM Ca²⁺ (G). (H) Schild plots quantifying the inhibition of ELIC mutants by ACh in the presence of Ca²⁺. EC₅₀ values were obtained from data shown in panels (E–G). Potencies of the antagonists (pA values) were obtained by linear regression; the intersection with the <i>x</i>-axis is indicated (•). The data presented in panels (B–C) and (E–G) are averages from at least 5 oocytes; errors are SD. The solid lines show fits to a Hill equation. Currents were recorded at −40 mV. A dose–response curve of WT in the absence of ACh and Ca²⁺ (dashed line) is shown for comparison.</p

Single-channel properties of α3β4 receptors expressed from extreme ratios in HEK293 cells.

<p>(A) Examples of outside-out currents elicited by 5 µM ACh, from cells transfected with an α3 to β4 cDNA ratio of 1∶9 (left) and 9∶1 (right). Patches were held at −100 mV. The histograms of fitted amplitudes corresponding to these recordings are shown in (B). These are fitted with Gaussian curves to give the peak current amplitudes and the areas under each curve (for these two patches the values are 2.7±0.3 pA, area 100% for 1∶9; 2.6±0.5 pA, area 27% and 3.9±0.4 pA, area 73% for 9∶1). The histogram on the left (C) shows the proportion of bursts (as % of the total number of bursts from all experiments, pooled) at each chord conductance for the two subunit ratios. The histogram on the right (D) shows the difference in the duration of the bursts to different conductances for the two ratios.</p