Thermodynamics and Kinetics of Ion Permeation in Wild-Type and Mutated Open Active Conformation of the Human α7 Nicotinic Receptor

Molecular studies of human pentameric ligand-gated ion channels (LGICs) expressed in neurons and at neuromuscular junctions are of utmost importance in the development of therapeutic strategies for neurological disorders. We focus here on the nicotinic acetylcholine receptor nAChR-α7, a homopentameric channel widely expressed in the human brain, with a proven role in a wide spectrum of disorders including schizophrenia and Alzheimer’s disease. By exploiting an all-atom structural model of the full (transmembrane and extracellular) protein in the open, agonist-bound conformation we recently developed, we evaluate the free energy and the mean first passage time of single-ion permeation using molecular dynamics simulations and the milestoning method with Voronoi tessellation. The results for the wild-type channel provide the first available mapping of the potential of mean force in the full-length α7 nAChR, reveal its expected cationic nature, and are in good agreement with simulation data for other channels of the LGIC family and with experimental data on nAChRs. We then investigate the role of a specific mutation directly related to ion selectivity in LGICs, the E-1′ → A-1′ substitution at the cytoplasmatic selectivity filter. We find that the mutation strongly affects sodium and chloride permeation in opposite directions, leading to a complete inversion of selectivity, at variance with the limited experimental results available that classify this mutant as cationic. We thus provide structural determinants for the observed cationic-to-anionic inversion, revealing a key role of the protonation state of residue rings far from the mutation, in the proximity of the hydrophobic channel gate.

For the wild type case, the system set up and the equilibration protocol of the protein/lipid bilayer/water system are the ones fully described in ref. 8 We remind here that protonation state of ionizable residues at physiological pH (7.4) was predicted by using the web-based implementation of a method for pKa calculations based on continuum electrostatic model 9 (http://biophysics.cs.vt.edu/H++) 10 ), by careful choice of the values for the dielectric constants of the protein domain interior/exterior, depending on the environment (solvent water or lipid bilayer) to which the domain is exposed. As result from these calculations, three out of five E-1 residues were found negatively charged, while the five E20' were found all protonated.
The total number of atoms in the wild type system is 142720 (26313 protein atoms including the five epibatidine molecules, 27360 water molecules, 255 POPC lipids, 77 Na + and 80 Cl − ions, corresponding to 100 mM solution, added to neutralize the net system charge). Starting from an equilibrated configuration of the wild type protein/bilayer/water system, we just replaced the five glutamate residues at position -1 in the protein with five alanine residues, by keeping unaltered the protonation state of the other residues. The total number of atoms in the new system is 142687 (26286 protein atoms plus five epibatidine molecules, 27357 water molecules, 255 POPC lipids, 77 Na + and 83 Cl − ions, corresponding to 100 mM solution, added to neutralize the new net system charge). The system was then equilibrated along a 0.5 µs simulation, with the same protocol as for the wild type system, 8 above described.

S2
As concerns PMF calculations in the wild type, the initial configurations for milestoning have been prepared by placing the target ion inside the protein along the z axis at distinct positions (the centers of the Voronoi cells) separated by 2Å starting from the intracellular mouth (corresponding to z =-19Å in our system) up to the extracellular mouth (corresponding to z =+93Å), see blue and red dots in Figure S1. Figure S1: Section of the system simulated (wild type case): two subunits only are shown, represented in cartoon, for sake of clarity. Dots indicate the centers of the Voronoi cells. Blue dots, cells 1-28 (from -19Å to +35Å); red dots, cells 29 to 57 (from +37Å to +93Å). The "sphere" adopted to ensure the single ion condition in the LBD is showed in blue. Lipids are in yellow (in lines representation); water in cyan (represented with points).
As for the E-1 A mutant, a representative structure was taken from the last snapshot after 0.5 µs MD simulation; the initial system configurations for milestoning calculations were prepared by placing the target ion along the z axis at distinct positions (the centers of Voronoi cells) separated by 2Å from the intracellular mouth (corresponding to z = -19Å) up to the TMD/LBD interface (corresponding toz = +35Å), see blue dots in Figure S1.
Estimate of the single-channel conductance were based on the single-ion PMF according to Roux et al, 11 by exploiting the PFM values in the TMD both in wild type and mutant.
To this aim, the diffusion coefficients of ions inside the TMD pore were set to half of the experimental bulk value (i.e 1.33 nm 2 /ns for sodium and 2.03 nm 2 /ns for chloride), as done S3 in Ref. 12 for simulations of Torpedo nAChrs.

Trajectory analysis Stability assessment
To assess the overall stability of the E-1 A mutant channel, we calculated: i) the Cα atoms    Figure S4, Figure S5 and Figure S6.

Electrostatic potential map
Protein electrostatic potential maps have been calculated using the PMEpot plugin in VMD. 17 The electrostatic potential is calculated using all the protein atoms, a three dimensional grid of 100 × 100 × 144 points (which ensures at least one grid point perÅ in each direction), and an Ewald factor of 0.25. The electrostatic potential generated by PMEpot is in units of kT/e; at T = 310 K, one PMEpot unit of electrostatic potential is equivalent to 27 mV. Results are shown in S7 and Figure S8. Figure S7: Protein electrostatic potential map, calculated on the structure averaged along a 200ns segment of the equilibrium trajectory of both wild type and E-1 A mutant. For sake of clarity, only two protein subunits (P3 and P5) are represented, in cartoon, colored according the electrostatic potential value. Upper panel: wild type; E-1 (E888) and E20 (E1565) are represented in vdw. Lower panel: E-1 A mutant. A-1 (A888) and E20 (E1565) are represented in vdw. One PMEpot unit of electrostatic potential, at T = 310K, is equivalent to 27mV. 17 Figure S8: As in S6, map calculated by averaging along the chloride trajectory restricted in the Voronoi cell centered at the V13 site.

E20 dynamics in the E-1 A structure
Side-chain dynamics of the E20 residues interacting with chloride has been investigated by monitoring the time evolution of the C-Cα-Cβ-Cγ torsion angle. Results are shown in Figure   S9. S10 Figure S9: Time evolution of the C-Cα-Cβ-Cγ angle of three E20 residues, along the trajectory restricted in the Voronoi cell at the V13 site. From top to bottom: E253, E909 and E1237, belonging to subunit P1, P3 and P4, respectively. S11