Structural basis for allosteric coupling at the membrane-protein interface in Gloeobacter violaceus ligand-gated ion channel (GLIC)

Abstract

Ligand binding at the extracellular domain of pentameric ligand-gated ion channels initiates a relay of conformational changes that culminates at the gate within the transmembrane domain. The interface between the two domains is a key structural entity that governs gating. Molecular events in signal transduction at the interface are poorly defined because of its intrinsically dynamic nature combined with functional modulation by membrane lipid and water vestibules. Here we used electron paramagnetic resonance spectroscopy to delineate protein motions underlying Gloeobacter violaceus ligand-gated ion channel gating in a membrane environment and report the interface conformation in the closed and the desensitized states. Extensive intrasubunit interactions were observed in the closed state that are weakened upon desensitization and replaced by newer intersubunit contacts. Gating involves major rearrangements of the interfacial loops, accompanied by reorganization of the protein-lipid-water interface. These structural changes may serve as targets for modulation of gating by lipids, alcohols, and amphipathic drug molecules.

Keywords: Cys-loop Receptors; Gating; Ion Channels; Membrane Reconstitution; Spectroscopy.

FIGURE 1.

Structural entities mediating allosteric communication at the intra- and intersubunit interfaces. A, overlay of ELIC and GLIC structures depicting the location of interfacial loops investigated in this study. ELIC and GLIC represent putative closed and open conformation, respectively. B, alignment of GLIC structure with the EM-based nAChR model in the unliganded state. The dashed box highlights the key ECD-TMD interface that shows pronounced differences between the channels.

FIGURE 2.

Biochemical and functional characterization of Cys mutants. A, representative gel filtration chromatograms of spin-labeled GLIC mutants showing the peak corresponding to the pentamer. Only mutants that displayed a monodisperse profile, corresponding to the pentameric channel population, were used for further analysis. B, macroscopic current measurements in response to pH jumps (from 8.0 to 2.5), at a −50 mV holding potential, for WT and representative Cys mutants. The current traces are normalized to their peak amplitudes. For the Phe-121 and Asp-154 mutants, the inset shows a 45-s current trace.

FIGURE 3.

pH-dependent changes in the EPR environmental parameter at the TMD loops. A, EPR spectra of representative residues within the pre-M1, M2-M3, and pre-M4 regions displaying changes in amplitude and line shapes in response to pH changes. Black and red traces were obtained from channels in the closed (pH 8.0) and in the desensitized conformations (pH 2.5), respectively. In each case, the spectra are normalized to the total number of spins. Arrows indicate positions with increased mobility in the desensitized conformation. B, plot of environmental parameter residues within each of the loops: mobility ΔH_o⁻¹ (top panel; gray, closed; black, desensitized) and accessibility (center and bottom panels; red, lipid accessibility (ΠO₂); blue, water accessibility (ΠNiEdda). The gray boxes highlight regions displaying prominent changes.

FIGURE 4.

Changes in dipolar spin-spin coupling at residues in M2 and the M2-M3 linker. EPR spectra of residues exhibiting spin-spin interactions. The red and black traces represent line shapes from the channel in the closed and pH-activated, desensitized conformations, respectively. The spectra marked by a dashed line were from underlabeled channels (in the presence of a diamagnetic label), and those marked by a solid line were from fully labeled channels. The inset shows an overlay of amplitude-normalized spectra. Broadening under fully labeled conditions is highlighted by arrows. The scan width is 150 G.

FIGURE 5.

Conformational rearrangements at the TMD loops during desensitization. Differences in the ΔH_o⁻¹, ΠO₂, and ΠNiEdda values for the closed and desensitized states mapped on the GLIC structure and color coded, with red denoting an increase and blue representing a decrease in the environmental parameter. Only two subunits are shown for clarity.

FIGURE 6.

Changes in intersubunit interaction during desentiziation. Differences in the ΔH_o⁻¹ (left panel) and ΠNiEdda (right panel) values for the pre-M1 segment and the M2-M3 linker in the closed and desensitized states. The Δ values were mapped on the GLIC structure, with red denoting an increase and blue representing a decrease in the environmental parameter. The indicated residues at the intersubunit interface display a decrease in dynamics/water accessibility. Only two subunits are shown for clarity.

FIGURE 7.

Extent of membrane insertion measured using DOGS-NTA[Ni(II)]. A, accessibility of representative residues within the indicated loops to membrane-incorporated DOGS-NTA[Ni(II)] in the closed and desensitized conformation. The arrows denote an increase or decrease in the ΠDOGS-NTA[Ni(II)]. B, ΔΠDOGS-NTA[Ni(II)] values mapped on to the GLIC structure and color-coded, with red denoting an increase and blue representing a decrease in the environmental parameter. Lipid molecules seen in GLIC structures are shown in orange. A schematic of DOGS-NTA[Ni(II)] in an extended conformation is also shown.

FIGURE 8.

pH-dependent changes in the EPR environmental parameter at the ECD loops. A, EPR spectra of representative residues within the β1-β2, β6-β7, and β8-β9 loops displaying changes in amplitude and line shapes in response to pH changes (black traces, closed conformation; red traces, desensitized conformation). In each case, the spectra are normalized to the total number of spins. B, plot of environmental parameters residues within each of the loops: Mobility ΔH_o⁻¹ (top panel; gray, closed; black, desensitized) and accessibility (center and bottom panels; red, lipid accessibility (ΠO₂); blue, water accessibility (ΠNiEdda). The gray boxes highlight regions displaying pronounced changes.

FIGURE 9.

Conformational rearrangements at the ECD loops during desensitization. Differences in the ΔH_o⁻¹, ΠO₂, and ΠNiEdda values for the closed and desensitized states mapped on the GLIC structure and color-coded, with red denoting an increase and blue representing a decrease in the environmental parameter.

FIGURE 10.

Dynamics of the β4-β5 loop. A, spectral line shapes in the closed (black) and desensitized (red) conformations. B, changes in the mobility and solvent accessibility parameters. C, differences in the EPR parameters mapped on to the GLIC structure.

FIGURE 11.

Schematic of structural changes at the ECD-TMD underlying GLIC gating. An EPR-based model predicting the structural changes at the interfacial loops during the transition to the desensitized state. Arrows indicate the putative direction of individual loop motion during transition to the desensitized state. The pH-dependent immobilization of the β9-β10 loop has been reported previously (9). Key conformational changes during desensitization involve weakening of the intrasubunit interactions involving the ECD loops (β1-β2 and β6-β7) and the TMD regions (pre-M1 region, the M2-M3 linker, and the C-terminal end of M4). As a consequence, there are new interactions at the intersubunit interface between the pre-M1 region and the M2-M3 linker from the adjacent subunit and also between loops β1-β2 and β8-β9.