Figure 1.
Ligand binding region of ELIC.
Left: ribbon representation of the ligand binding domain of ELIC. The subunits contributing to the principal and complementary side of the binding region are colored in green and orange, respectively. The binding site is marked by a grey box. Right: zoom into the binding site. The protein is shown as Cα-trace with residues lining the binding pocket shown as stick model. The residues and secondary structure elements are labeled. Structures displayed in Figures 1, 3, and 5 were prepared with DINO (www.dino3d.org).
Figure 2.
Current response of ELIC to different ligands.
The chemical structures of the ligands are shown above the traces. Currents were recorded from oocytes at −60 mV with the two electrode voltage clamp technique. Top left: the response to 2 mM cysteamine is shown for water-injected control oocytes and for oocytes expressing ELIC. The application of the ligand is indicated (bar, *). All other traces show the response to agonists in comparison to the response to cysteamine. Agonists were applied to the outside in a concentration of 10 mM. The activation by cysteamine (grey bar, 2 mM) is followed by the application of a different agonist (black bar, *, 10 mM) and another activation by cysteamine (grey bar, 2 mM). Ligands are grouped according to their chemical properties and length of the aliphatic chains.
Figure 3.
(A) The current response upon application and washout of cysteamine was recorded at −60 mV with the two-electrode voltage clamp technique. The application of cysteamine at the respective concentration is indicated above (black bar). The relative open probability (I/Imax) plotted as a function of the ligand concentration is shown below. The currents were normalized to the maximum at saturating ligand concentration (i.e., 5 mM). The average of 10 oocytes and their standard deviations are shown. The solid line shows a fit to a Hill equation with a coefficient of 2.7. (B) Activation and deactivation kinetics. Macroscopic currents from a membrane patch in the outside-out configuration were recorded at −60 mV in response to a fast exchange into solutions containing 5 mM cysteamine. The fit of the current increase upon application of the ligand and the decrease upon washout to a single exponential function is shown below. (C) Dose response curves for the activation of ELIC by different agonists. Currents were recorded with the two electrode voltage clamp technique at −60 mV. The solid lines show fits to a Hill equation. The dose response curve for cysteamine (black, dashed line) is shown for comparison. (D) Dose response curves for the activation of ELIC by diamines. Currents were recorded with the two electrode voltage clamp technique at −60 mV and pH 8. The solid lines show fits to a Hill equation. The red trace (putrescine*) shows a fit to the fraction of putrescine at pH 8 that carries a single positive charge. The dose response curve for cysteamine (black, dashed line, measured at pH 7) is shown for comparison.
Table 1.
Dose-response relationships of different agonists of ELIC.
Figure 4.
Ligand binding and mutagenesis.
(A) Anomalous difference map of ELIC in complex with bromopropylamine. One of two pentamers of ELIC in the asymmetric unit of the crystal and a zoom into a single binding pocket are shown. The subunits of the principal and complementary side are colored in green and orange, respectively. The anomalous difference map calculated at 5.0 Å and contoured at 4.5 σ is shown as red mesh. The ligand binding region is indicated by a grey box. A model of bromopropylamine is shown in ball and stick representation. (B) Activation of ligand binding site mutants by cysteamine. Dose response curves from currents recorded with the two-electrode voltage clamp technique are shown. The solid lines show fits to a Hill equation. (C) Activation of ligand binding site mutants by aminopropanol. The solid lines show fits to a Hill equation, and dashed lines in the same color show the activation of the respective mutants by cysteamine. (D) Structure of the mutant F246A. Left: Superposition of Cα traces of the pore region of WT (red) and the mutant F246A (green). The view is from within the membrane; the front subunit is removed for clarity. Right: 2Fo-Fc electron density (calculated at 3.3 Å and contoured at 1 σ, blue mesh) of the pore region of the mutant F246A superimposed on the refined structure (green). The WT structure (red is shown for comparison). The view is from the extracellular side. The missing electron density for the aromatic side chains is apparent. (E) Activation of the pore mutant F246A by cysteamine. Dose response curves from currents recorded with the two-electrode voltage clamp technique are shown. The solid lines show fits to a Hill equation. The WT is shown for comparison.
Table 2.
Dose-response relationship of activation of ELIC point mutants.
Table 3.
Data collection and refinement statistics.
Figure 5.
Single channel recording of ELIC.
(A) Patch clamp recordings of ELIC expressed in Xenopus oocytes in the outside-out configuration at −60 mV. The agonist (5 mM cysteamine) was applied by a fast solution exchange (black bar above). The decrease of channel activity over time is due to desensitization. The magnified trace shows activity of a single channel. Recordings were filtered at 1 kHz. (B) Current-voltage relationships recorded from single channels of ELIC expressed in Xenopus oocytes in a patch clamp experiment (blue) and of reconstituted protein in artificial lipid bilayers (red). The errors are standard deviations (s.d.) obtained from fits to current amplitude histograms. (C) Current trace from a planar lipid bilayer containing at least two channels recorded at −100 mV in the presence of 2 mM cysteamine (recordings were filtered at 200 Hz). (D) Current voltage relationships of ELIC in asymmetric concentrations of NaCl measured in the presence of 2 mM cysteamine. The currents reverse at the Nernst potential of Na+. The compartment with lower ion concentration corresponds to the “extracellular side.” Data shown in panels D–G and I were measured from single channels in planar lipid bilayers. The errors are s.d. obtained from fits to current amplitude histograms. Channels were activated by addition of 2 mM cysteamine to both compartments unless stated differently. (E) Current voltage relationships in asymmetric conditions containing equivalent amounts of different monovalent salts. (F) Current voltage relationships of single channel currents at different concentrations of NaCl. (G) Current voltage relationships in asymmetric salt conditions. The “extracellular side” contains 150 mM NaCl, and the “intracellular side” contains different concentrations of CaCl2 (25–150 mM). Channels were activated by addition of 2 mM cysteamine to the “extracellular side” only. Data from symmetric concentrations of NaCl (150 mM, black) are shown for comparison. (H) Concentration dependence of the single channel conductance of Na+ and Ca2+ currents. (I) Current-voltage relationships of the mutant E229A in symmetric concentrations of NaCl. Channels were activated by addition of 2 mM cysteamine to the “extracellular side.” Data from the WT channel (green) are shown for comparison.
Figure 6.
Biology and hypothetical mechanisms.
(A) Localization of ELIC on the genome of Erwinia chrysanthemi. Open reading frames residing on the same operon and their respective positions on the gene are shown. The arrows indicate the direction of transcription. The annotation is based on sequence homology. The reaction catalyzed by the annotated glutamate decarboxylase (AA Decarboxylase) is shown above. (B) Potential conformational transitions during ligand activation. Upon ligand binding the channel changes from a resting state (c) to an open state (o) followed by a transition to a desensitized state (d). (Top) Comparison of the binding pocket of ELIC in a non-conducting conformation with a hypothetical structure of the region in a conducting state. The model of the conducting state (red) was generated by independent counterclockwise rigid-body rotations of the ligand binding domain of ELIC (green) by about 12° around the axis indicated in the figure. The rotation axis was obtained from a least square fit of conserved regions of the ligand binding domain of ELIC on the equivalent regions of GLIC. The binding sites of both states are shown. The ligand binding pockets are displayed as grey mesh. A model of cysteamine (shown as CPK representation) was placed into the binding site in a similar binding mode as observed in the structure of ELIC in complex with bromopropylamine. (Bottom) Hypothetical structure of the pore region in a conducting conformation. The conducting state of the pore region (red) was constructed by a rigid body rotation of the α2-α3 helix pair of a subunit of ELIC (green) by 12° in a counterclockwise direction around an axis that is indicated in the figure. The model of the pore region of this conformation is displayed as Cα trace. Side chains of pore forming helices are shown as sticks. The solvent accessible surface is shown as grey mesh. The front subunit is removed for clarity. The pore radii of GLIC (green, with the side chains of Glu 221 pointing away from the channel axis) and the hypothetical conducting conformation of ELIC as calculated with the program HOLE [61] are shown (the cytoplasm is on the left side).