Structural sensitivity of a prokaryotic pentameric ligand-gated ion channel to its membrane environment

Abstract

Although the activity of the nicotinic acetylcholine receptor (nAChR) is exquisitely sensitive to its membrane environment, the underlying mechanisms remain poorly defined. The homologous prokaryotic pentameric ligand-gated ion channel, Gloebacter ligand-gated ion channel (GLIC), represents an excellent model for probing the molecular basis of nAChR sensitivity because of its high structural homology, relative ease of expression, and amenability to crystallographic analysis. We show here that membrane-reconstituted GLIC exhibits structural and biophysical properties similar to those of the membrane-reconstituted nAChR, although GLIC is substantially more thermally stable. GLIC, however, does not possess the same exquisite lipid sensitivity. In particular, GLIC does not exhibit the same propensity to adopt an uncoupled conformation where agonist binding is uncoupled from channel gating. Structural comparisons provide insight into the chemical features that may predispose the nAChR to the formation of an uncoupled state.

FIGURE 1.

The structures of GLIC (A, Protein Data Bank code 3EAM) and the Torpedo nAChR (B, Protein Data Bank code 2BG9). Both structures are side views from within the plane of the membrane. Coloring highlights the domain structure: the extracellular agonist-binding domain (ABD) in red, the transmembrane domain (TMD) in blue, and the cytoplasmic domain (CD) in green.

FIGURE 2.

Structural comparisons of membrane-reconstituted GLIC and the nAChR as probed by infrared spectroscopy. A, infrared spectra of aso-GLIC recorded after gentle drying from ¹H₂O buffer (solid black line) and immediately after addition of ²H₂O (dashed gray line). Note the immediate changes in amide I band shape (1700–1600 cm⁻¹) and the immediate decrease in amide II band intensity (1547 cm⁻¹), both indicative of the rapid peptide N-¹H/N-²H exchange of solvent-exposed peptide hydrogens. Similar spectral effects are observed for other Cys-loop receptors (supplemental Fig. S2). B, infrared spectra recorded after 72 h of equilibration in ²H₂O at 4 °C from aso-GLIC (spectrum i), EcoLip-GLIC (spectrum ii), PC-GLIC (spectrum iii), aso-nAChR (spectrum iv), and PC-nAChR (spectrum v). The left column shows the secondary structure-sensitive amide I band both before (gray traces) and after resolution enhancement (black traces) (intensity scaling arbitrary). The right column shows the amide II band in each spectrum. The relative intensity of the amide II vibration is best assessed relative to the intensity of the adjacent broad peak between 1560 and 1600 cm⁻¹, because of aspartic and glutamic acid residues. All of the presented spectra are the averages of several spectra recorded from at least two different purification/reconstitutions.

FIGURE 3.

Representative thermal denaturation curves for GLIC (black lines) and the nAChR (gray lines) in different membranes. The denaturation curves are for aso-GLIC (▴), EcoLip-GLIC (●), PC-GLIC (□), aso-nAChR (▵), PC/PA/Chol-nAChR (♦), and PC-nAChR (■). Each curve was fit with a Boltzmann sigmoid from which T_d and Boltzmann slope were calculated using GraphPad Prism software (Table 1) (see supplemental materials and Ref. 11). The Boltzmann slope decreases with increasing cooperativity of unfolding.

FIGURE 4.

The Carb-induced response of membrane-reconstituted nAChR after injection into and consequent fusion with the plasma membrane of Xenopus oocytes. A, currents induced by 500 μm Carb were measured at −20 mV holding potential from oocytes injected with aso-nAChR (left trace) and PC-nAChR (right trace). B, a bar graph comparing maximal recorded currents, each normalized to the number of [¹²⁵I]α-bungarotoxin (BTX)-binding sites, induced by 300 μm acetylcholine from individual oocytes microinjected with 125 ng of affinity-purified Torpedo nAChR protein reconstituted in either PC/PA/Chol (3:1:1 molar ratio; 0.124 ± 0.061 μA/[¹²⁵I]α-bungarotoxin-binding sites/oocyte; n = 5) or PC lipid vesicles (0.0034 ± 0.0006 μA/[¹²⁵I]α-bungarotoxin-binding sites/oocyte; n = 6).

FIGURE 5.

The proton-induced response of membrane-reconstituted GLIC after injection into and consequent fusion with the plasma membrane of Xenopus oocytes. A, electrophysiology recordings in response to pH jumps from uninjected oocytes and oocytes injected with aso-GLIC, EcoLip-GLIC, and PC-GLIC. The dose-response curves were measured at a relatively low membrane potential of −20 mV, because this seemed to generate more stable base lines. An electrical response to pH 4.0 showing the effect of 150 μm amantadine (gray bar) is shown on the right (scaling arbitrary). The latter were performed at a membrane potential of −60 mV. B, peak current achieved upon exposure of oocytes injected with the indicated reconstituted membranes at pH 3.5. The reported values are the averages ± standard deviation from five recordings performed on five difference oocytes. C, comparison of the dose-response curves obtained from oocytes injected with the membrane-reconstituted GLIC or GLIC mRNA. The dose-response curves from oocytes injected with membrane-reconstituted GLIC were normalized assuming a pH₅₀ of 2.90 according to Velisetty et al. (51).

FIGURE 6.

Aromatic-aromatic interactions may dictate the propensity of a pLGIC to adopt a lipid-dependent uncoupled conformation. Shown is a comparison of the aromatic residues located in M1, M3, and M4 of a single subunit transmembrane domain for GLIC (A) and the α-subunit of the nAChR (B). Both a side view of the transmembrane domain of each subunit and a top view looking down at the bilayer surface are shown. Note that the side view orientations of the two transmembrane domains have been rotated 180° about the long axis of each molecule relative to the side view orientation shown in the schematic diagram of uncoupling in supplemental Fig. S6, because this presents a clearer view of the aromatic-aromatic interactions at the interface between M4 and M1+M3.