Intramembrane proton binding site linked to activation of bacterial pentameric ion channel

Abstract

Prokaryotic orthologs of eukaryotic Cys-loop receptor channels recently emerged as structural and mechanistic surrogates to investigate this superfamily of intercellular signaling proteins. Here, we examine proton activation of the prokaryotic ortholog GLIC using patch clamp electrophysiology, mutagenesis, and molecular dynamics (MD) simulations. Whole-cell current recordings from human embryonic kidney (HEK) 293 cells expressing GLIC show half-maximal activation at pH 6, close to the pK(a) of histidine, implicating the three native His residues in proton sensing linked to activation. The mutation H235F abolishes proton activation, H277Y is without effect, and all nine mutations of His-127 prevent expression on the cell surface. In the GLIC crystal structure, His-235 on transmembrane (TM) α-helix 2, hydrogen bonds to the main chain carbonyl oxygen of Ile-259 on TM α-helix 3. MD simulations show that when His-235 is protonated, the hydrogen bond persists, and the channel remains in the open conformation, whereas when His-235 is deprotonated, the hydrogen bond dissociates, and the channel closes. Mutations of the proximal Tyr-263, which also links TM α-helices 2 and 3 via a hydrogen bond, alter proton sensitivity over a 1.5 pH unit range. MD simulations show that mutations of Tyr-263 alter the hydrogen bonding capacity of His-235. The overall findings show that His-235 in the TM region of GLIC is a novel proton binding site linked to channel activation.

FIGURE 1.

Ionic currents evoked by step changes in pH for HEK 293 cells expressing GLIC and mutants of the three intrinsic His residues. A, crystal structure of GLIC (7) (PDB code 3EAM) with the three His residues shown in space-filling representation. B, ionic currents versus time evoked by the indicated step changes in pH for mock-transfected cells, wild type GLIC, and the indicated His mutants. Currents with fast response shown in mock-transfected cells are from endogenous pH-sensitive channels.

FIGURE 2.

Cell surface expression of GLIC in which loop C was replaced with that from the human α7 AChR. Shown is [¹²⁵I]α-bungarotoxin bound to intact cells expressing GLIC with loop C modified and the indicated mutants. Inset, pH-activated currents for wild type GLIC and GLIC with loop C modified.

FIGURE 3.

Stereo view of the interhelical cleft separating TMs 2 and 3 obtained from the GLIC crystal structure (7). Key residues and hydrogen bonds are indicated.

FIGURE 4.

MD simulations of GLIC with His-235 unprotonated and protonated. The left panel shows the hydrogen bond between Nϵ of His-235 and the main chain oxygen atom of Ile-259. The right panels show the interatomic distance versus simulation time for each condition. Black traces indicate the interatomic distances averaged over 40 ps, whereas the light traces show distances measured every 2 ps.

FIGURE 5.

Pore radius versus simulation time for the indicated GLIC structures. For each panel, pore radius is indicated by color along an axis passing through the center of the pore (y axis) as a function of simulation time (x axis). Pore radii were calculated using the program HOLE 2.0 (31).

FIGURE 6.

Upper panel, top views of the GLIC structures from the protonated (left) and unprotonated (right) simulations, with Ile-233 at the narrowest constriction of TM 2 highlighted in surface rendering and in different colors in each subunit. Lower panel, cross-sectional view of time-averaged cation distributions. In both structures, cations are concentrated (red) in the upper half of the channel. With His-235 protonated, there is an open pathway with increased cation density, whereas with His-235, the unprotonated the pathway is occluded.

FIGURE 7.

pH activated ionic currents for mutations of Tyr-263. A, ionic currents versus time in response to the indicated step changes in pH for wild type GLIC and the indicated mutants of Tyr-263. B, relationships between ionic current and pH for GLIC and the indicated mutants. Smooth curves are fits of the Hill equation to the data with the following parameters: for wild type GLIC the EC₅₀ = 1.09 × 10⁻⁶ m (pH = 5.96), and the Hill coefficient n_H = 2.20 + 0.15 (n = 5); for Y263T the EC₅₀ = 1.38 × 10⁻⁷ m (pH = 6.86) and n_H = 2.9 + 0.44 (n = 3); for Y263F the EC₅₀ = 5.088 × 10⁻⁶ m (pH = 5.3) and n_H = 1.36 + 0.15 (n = 3).

FIGURE 8.

Stability of the hydrogen bond formed by His-235 obtained from MD simulations of GLIC and virtual mutations of Tyr-263. Panel B plots of the probability of the hydrogen bond between the Nϵ atom of His-235 and the main chain oxygen atom of Ile-259 versus the hydrogen bond distance. Panel A shows interhelical hydrogen bonds for the Y263T mutant.