Drying transition in the hydrophobic gate of the GLIC channel blocks ion conduction

Abstract

The theoretical prediction of water drying transitions near nonpolar surfaces has stimulated an intensive search for biological processes exploiting this extreme form of hydrophobicity. Here we quantitatively demonstrate that drying of a hydrophobic constriction is the major determinant of ion conductance in the GLIC pentameric ion channel. Molecular-dynamics simulations show that in the closed state, the channel conductance is ∼12 orders-of-magnitude lower than in the open state. This large drop in conductance is remarkable because even in the functionally closed conformation the pore constriction remains wide enough for the passage of sodium ions, aided by a continuous bridge of ∼12 water molecules. However, we find that the free energy cost of hydrating the hydrophobic gate is large, accounting almost entirely for the energetic barrier blocking ion passage. The free energies of transferring a sodium ion into a prehydrated gate in functionally closed and open states differ by only 1.2 kcal/mol, compared to an 11 kcal/mol difference in the costs of hydrating the hydrophobic gate. Conversely, ion desolvation effects play only minor roles in GLIC ion channel gating. Our simulations help rationalize experiments probing the gating kinetics of the nicotinic acetylcholine receptor in response to mutations of pore-lining residues. The molecular character and phase behavior of water should thus be included in quantitative descriptions of ion channel gating.

Figure 1

C_α traces of the M2 and M3 helices in the transmembrane domain. The crystal structures of ELIC (25), GLIC (26), and the computational model (18) of the closed GLIC channel in our simulations are superimposed. (Dashed line) Symmetry axis of the protein.

Figure 2

Free energy of hydration and ion translocation in GLIC. (a) Free energy $G\left(\hat{N}\right)$ as a function of the coarse-grained water number $\hat{N}$ inside the hydrophobic gate. For the open conformation, the free energy $G\left(\hat{N}\right)=-k_{\text{B}}T\text{ln}p\left(\hat{N}\right)$ was obtained directly from the equilibrium probability distribution $p\left(\hat{N}\right)$ in a 40-ns simulation (18). For the closed conformation, umbrella sampling was used (see Methods). (b) Free energy G(z) as a function of the z-coordinate of a Na⁺ ion, for the open and closed conformations. The extracellular side is at positive z values. (Two vertical dashed lines) Boundaries of the hydrophobic gate. (c) Probability distribution (heat map) and average (solid curve) of water occupancy $\hat{N}$ with a Na⁺ ion restrained to different z-positions in closed-state simulations. (Dashed arrow) Plateau value $\hat{N}$ ≈ 12 when the ion is inside the hydrophobic gate.

Figure 3

Snapshots of cuts through the GLIC pore interior, with the extracellular side up. Water molecules within and immediately outside the hydrophobic gate region are shown (51). (Arrows on the right) Approximate positions of the Ile²³² and Ile²³⁹ rings. Free energy differences between the states are given in kcal/mol. (a) Closed conformation without restraint on the water number or ion position (18). (b) Closed conformation with a water occupancy of $\hat{N}\approx 12$, corresponding to the average value of $\hat{N}$ when an ion is restrained to the center of the gate region. (c) Closed conformation with a Na⁺ ion restrained to the gate region. (d) Open conformation with a Na⁺ ion restrained to the gate region.

Figure 4

Free energy G(z) of the ion (left scale) and the average water occupancy (right scale) as a function of the ion position for the closed conformation, as in Fig. 2, b and c, respectively.