Signal Transduction at the Domain Interface of Prokaryotic Pentameric Ligand-Gated Ion Channels

Abstract

Pentameric ligand-gated ion channels are activated by the binding of agonists to a site distant from the ion conduction path. These membrane proteins consist of distinct ligand-binding and pore domains that interact via an extended interface. Here, we have investigated the role of residues at this interface for channel activation to define critical interactions that couple conformational changes between the two structural units. By characterizing point mutants of the prokaryotic channels ELIC and GLIC by electrophysiology, X-ray crystallography and isothermal titration calorimetry, we have identified conserved residues that, upon mutation, apparently prevent activation but not ligand binding. The positions of nonactivating mutants cluster at a loop within the extracellular domain connecting β-strands 6 and 7 and at a loop joining the pore-forming helix M2 with M3 where they contribute to a densely packed core of the protein. An ionic interaction in the extracellular domain between the turn connecting β-strands 1 and 2 and a residue at the end of β-strand 10 stabilizes a state of the receptor with high affinity for agonists, whereas contacts of this turn to a conserved proline residue in the M2-M3 loop appear to be less important than previously anticipated. When mapping residues with strong functional phenotype on different channel structures, mutual distances are closer in conducting than in nonconducting conformations, consistent with a potential role of contacts in the stabilization of the open state. Our study has revealed a pattern of interactions that are crucial for the relay of conformational changes from the extracellular domain to the pore region of prokaryotic pentameric ligand-gated ion channels. Due to the strong conservation of the interface, these results are relevant for the entire family.

Fig 1. Domain Interface of pLGICs.

(A) Aligned sequences of the domain interface of GLIC and ELIC with residue numbering shown above and below, respectively. Secondary structure elements are indicated. Identical residues are colored in blue. Residues mutated in this study in either protein are highlighted in yellow. (B) Domain interface of ELIC (Protein Data Bank entry 2VL0). The protein is displayed as Cα-trace, mutated regions are shown in unique colors, the views are indicated. Left: View at the interface from the outside. Right: View from the pore region on two adjacent subunits. (C) Domain interface of GLIC (Protein Data Bank entry 3EHZ). The protein is displayed as Cα-trace, with mutated regions shown in unique colors. The views are as in B.

Fig 2. Nonactivating mutations at the domain interface.

Cα-trace of parts of the domain interface in a single subunit of (A), ELIC and (B), GLIC with sidechains of selected nonactivating mutants shown as CPK models. The views are from within the pore (as in Fig 1B, right panel). Residues in ELIC for which alanine mutants were characterized in detail (Phe116 and Tyr258) and the corresponding residues in GLIC (Phe115 and Tyr250) are colored in red. (C–D), Surface expression of nonactivating mutants of (C), ELIC and (D), GLIC. Data show averages of 7–14 different oocytes and are normalized to wild type (WT). Background of noninjected (non inj.) oocytes was not subtracted. Errors are standard error of the mean (SEM). (E–F), Current response of a representative oocyte at high agonist concentration that was subsequently used for the analysis of surface expression. Currents were recorded at −40 mV. (E) Response in ELIC after application of 25 mM cysteamine. (F) Response in GLIC after change to pH 4. Application of agonist is indicated by a red bar. (See S1 Data for the raw data used to generate plots shown in panels C and D).

Fig 3. Characterization of two nonactivating mutants of ELIC.

(A) Refined structure of the ELIC mutant F116A. Left, Cα-trace of part of a single subunit of F116A (grey) showing the domain interface superimposed on ELIC WT (orange). Right, interface of F116A with sidechains of selected residues shown as CPK models. View and selection are as in Fig 2A. (B) Analogous representation of the refined structure of the mutant Y258A. In A and B, the site of mutation is indicated by an asterisk. (C) Macroscopic currents recorded from representative patches of ELIC WT upon fast application and washout of agonist. Left, current response of a membrane patch of X. laevis oocytes expressing ELIC upon application of 20 mM cysteamine. Right, current recorded from a membrane patch of ELIC expressed in human embryonic kidney 293 (HEK293) cells upon application of 25 mM propylamine. The difference in magnitude of the currents is due to the higher expression of ELIC in HEK293 cells. (D) Representative current response of a membrane patch of the ELIC mutant F116A expressed in X. laevis oocytes upon application of 20 mM cysteamine. (E) Representative current response of a membrane patch of the ELIC mutant Y258A expressed in HEK293 cells upon application of 25 mM propylamine. C–E, Currents were recorded from excised patches in the outside-out configuration at a potential of −50 mV for HEK cell and −80 mV for X. laevis oocyte patches. Application of agonist by fast solution exchange is indicated by a black bar. (F–I), ITC experiments. Agonist and antagonist binding to (F), ELIC WT, (G), the ligand binding site mutant R91A, the double mutants (H), R91A/F116A and (I), R91A/Y258A. Graphs show a fit of the integrated and corrected heat upon addition of the agonist propylamine (left) and the antagonist acetylcholine (right) to a binding isotherm (red line). Experiments were repeated twice with similar results. (See S1 Data for the raw data used to generate plots shown in panels F–I).

Fig 4. ELIC mutant with strongly increased potency for the agonist.

Structure of the interaction region between the β1-β2 turn, a conserved arginine the end of β-10 and the β6-β7 loop in (A), ELIC and (B), GLIC. The view is from the extracellular side. Side-chains of selected residues forming a branched salt bridge in GLIC and their equivalent residues in ELIC are shown as CPK models. (C) Dose-response relationships of X. laevis oocytes expressing the ELIC mutants T28A and T28D measured by two-electrode voltage clamp electrophysiology. Current response of WT is shown for comparison. All currents were recorded at an outside Ca²⁺ concentration of 0.5 mM. Panels show averages of 4–6 independent measurements, solid lines show a fit to a Hill equation, errors are standard deviation (SD). (D) Agonist and antagonist binding to the ELIC mutant T28D as determined by ITC. Graphs show a fit of the integrated and corrected heat upon addition of the agonist propylamine (left) and the antagonist acetylcholine (right) to a binding isotherm (red line). (E) Patch clamp recording of the ELIC mutant T28D expressed in X. laevis oocytes. Currents were measured from excised patches in the outside-out configuration at −80 mV. Application of 20 mM cysteamine by fast solution exchange is indicated by a black bar. The recording shows considerable basal activity in the absence of the ligand. (See S1 Data for the raw data used to generate plots shown in panels C and D).

Fig 5. Interaction between the β1-β2 turn and the pore domain.

Relationship between the residue at the tip of the β1-β2 turn and a conserved proline at the M2-M3 loop in (A), ELIC and (B), GLIC. The view is parallel to the membrane plane. Side chains of selected residues are shown as CPK models. (C–F), Dose-response relationships of X. laevis oocytes expressing mutants of either ELIC or GLIC measured by two-electrode voltage clamp electrophysiology. Current response of the respective WT is shown for comparison. For ELIC, currents were recorded at an outside Ca²⁺ concentration of 0.5 mM. Panels show averages of 3–5 independent measurements, solid lines are a fit to a Hill equation, errors are SD. Mutants of the β1-β2 turn: (C), ELIC mutant L29A and the channel carrying a deletion of Leu29 (ΔL29). (D), GLIC mutant K32A. Mutants of the M2-M3 loop: (E), ELIC mutants P254A and P254G. (F), GLIC mutants P246A and P246G. For ELIC P254G, ligand was not removed following application due to the very slow deactivation of the channels. (G) Refined structure of the ELIC mutant P254G. Left, Cα-trace of part of a single subunit of P254G (grey) showing the domain interface superimposed on ELIC WT (orange). An asterisk marks the site of mutation. Right, structure of the M2-M3 loop of the mutant P254G and of WT with side chains of R255A and E155 from the adjacent subunit shown as CPK model. (H), Structure of the GLIC mutant P246G. Left, Cα-trace of part of a single subunit of P246G (grey) showing the domain interface and part of the pore region superimposed on GLIC WT (green). An asterisk marks the site of mutation. Right, Pore radius of GLIC (blue) and P246G (red) along the channel axis. The membrane boundary is indicated. (I) Macroscopic currents recorded from representative patches of the ELIC mutant P254G (left) and the double mutant P254G/R255A (right) upon fast application and washout of agonist. Currents were recorded from membrane patches of the respective ELIC mutants expressed in HEK293 cells in response to application of 25 mM propylamine. The difference in the deactivation rate in both mutants is apparent. (See S1 Data for the raw data used to generate plots shown in panels C-F).

Fig 6. Role of the domain interface in channel activation.

Interactions between residues at the domain interface in different channel conformations. (A) Sequence alignment of regions of the domain interface. Selected residues and their interactions are highlighted and schematically depicted above. Solid lines indicate interactions that are present in all conformations and dashed line interactions that are only formed in the presumably open GLIC-like conformation. Numbering below the sequences indicates the position of residues in the pore. Sequences are GLIC (UniProt: Q7NDN8), ELIC (GB: WP_013319743), GluCl (GB: AAA50785.1), GlyR (GB: NP_571477.1), Gaba_A (GB: M82919), 5-HT3 (GB: NM_001099644.1), AChRα (UniProt: P04756), AChRα7 (UniProt: P49582). Cα-trace of the interface of a single subunit of (B) GLIC in a presumably open conformation (GLIC-like), (C) GLIC at pH 7.0 (locally-closed), (D) ELIC (ELIC-like). The view is from within the membrane (left) and from the extracellular side (right). The pore domain is colored in blue and the extracellular domain in green. The positions of selected residues whose mutation exerts a strong effect on activation in ELIC and GLIC are shown as spheres. The residue at the tip of the β1-β2 turn and a conserved proline in the M2-M3 loop are colored in red. Selected interactions (as in A) are indicated by dashed lines.