Mechanism of Calcium Permeation in a Glutamate Receptor Ion Channel

Abstract

The α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPARs) are neurotransmitter-activated cation channels ubiquitously expressed in vertebrate brains. The regulation of calcium flux through the channel pore by RNA-editing is linked to synaptic plasticity while excessive calcium influx poses a risk for neurodegeneration. Unfortunately, the molecular mechanisms underlying this key process are mostly unknown. Here, we investigated calcium conduction in calcium-permeable AMPAR using Molecular Dynamics (MD) simulations with recently introduced multisite force-field parameters for Ca²⁺. Our calculations are consistent with experiment and explain the distinct calcium permeability in different RNA-edited forms of GluA2. For one of the identified metal binding sites, multiscale Quantum Mechanics/Molecular Mechanics (QM/MM) simulations further validated the results from MD and revealed small but reproducible charge transfer between the metal ion and its first solvation shell. In addition, the ion occupancy derived from MD simulations independently reproduced the Ca²⁺ binding profile in an X-ray structure of an NaK channel mimicking the AMPAR selectivity filter. This integrated study comprising X-ray crystallography, multisite MD, and multiscale QM/MM simulations provides unprecedented insights into Ca²⁺ permeation mechanisms in AMPARs, and paves the way for studying other biological processes in which Ca²⁺ plays a pivotal role.

Figure 1

(A) AMPARs (PDB ID: 5WEO) consist of an Amino Terminal Domain (ATD), a Ligand Binding Domain (LBD), and a Transmembrane Domain (TMD). (B) The TMD and linkers to the LBD (cyan box) were included in the computational electrophysiology simulations, where the transmembrane potential was generated by introducing an ion imbalance between compartment A and B. (C) A snapshot of GluA2(Q) selected from the MD simulations showing the selectivity filter region and coordinating Ca²⁺ ions (orange spheres). Water molecules are represented by lines, protein in cartoon with key residues in sticks. For better visibility, only two opposing subunits are shown. (D) A selected QM/MM snapshot. Atoms that are included in the QM partition (Ca_aq²⁺) are shown in ball and stick with their electron density (isovalue 0.1 e/a₀³) in gray wireframe.

Figure 2

(A) Representative traces of Ca²⁺ passing through the SF of the GluA2(Q) channel pore during a “high conductive” (top) and a “low conductive” MD simulation run (bottom). The cross-section of the GluA2 transmembrane domain is shown in the second column together with the two-dimensional Ca²⁺ occupancy derived from MD. The latter is plotted on a logarithmic scale as concentration, and linearly as free energy. (B) Selected snapshots of “low” and “high” conductive MD simulations revealing the major Ca²⁺ binding sites within and above the SF. In the selected “high conductive” snapshot, only Ca²⁺ at sites 2 and 4 are present simultaneously, while two modeled ions at sites 1 and 3 are displayed in transparent spheres.

Figure 3

(A) Number of coordinated water and protein residues during calcium permeation through the channel pore for high and low conductive runs. (B) Calcium–oxygen radial distribution function (red) and running coordination number (gray) at z = 8 Å. Solid/dashed lines indicate results from QM/MM/MD, respectively.

Figure 4

(A) Integrated charge transfer between the calcium ion and its first hydration sphere evaluated for 82 snapshots on the B3LYP level and grouped by element. (B) Electron density difference Δϱ between the presence and absence of Ca²⁺ for one QM/MM snapshot. Blue/green surfaces represent an isovalue of +0.005/ – 0.005 e/a₀³, respectively. (C) Maximally localized Wannier functions of coordinated water molecules (gray dots) represent oxygen lone pairs and O–H bonds.

Figure 5

(A) Relative one-dimensional ion occupancy in the SF of NaK C-DI derived from Ca²⁺ MD simulations (orange), performed with the multisite Ca²⁺ model compatible with CHARMM (this work) and K⁺ MD simulations (cyan), with CHARMM. The origin is set to the hydroxy group of T63. (B) The 2F_o–F_c electron density maps (blue mesh, contoured at 1σ) around Ca²⁺ (orange), Rb⁺ (cyan) and water molecules (red) along the ion channel path of NaK C-DI together with the anomalous difference density of Rb⁺ (cyan mesh), contoured at 3σ.