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. 2023 Apr 4;122(7):1158-1167.
doi: 10.1016/j.bpj.2023.02.005. Epub 2023 Feb 10.

Inner pore hydration free energy controls the activation of big potassium channels

Erik B Nordquist  1 , Zhiguang Jia  1 , Jianhan Chen  2
Affiliations

Inner pore hydration free energy controls the activation of big potassium channels

Erik B Nordquist et al. Biophys J. .

Abstract

Hydrophobic gating is an emerging mechanism in regulation of protein ion channels where the pore remains physically open but becomes dewetted to block ion permeation. Atomistic molecular dynamics simulations have played a crucial role in understanding hydrophobic gating by providing the molecular details to complement mutagenesis and structural studies. However, existing studies rely on direct simulations and do not quantitatively describe how the sequence and structural changes may control the delicate liquid-vapor equilibrium of confined water in the pore of the channel protein. To address this limitation, we explore two enhanced sampling methods, namely metadynamics and umbrella sampling, to derive free-energy profiles of pore hydration in both the closed and open states of big potassium (BK) channels, which are important in cardiovascular and neural systems. It was found that metadynamics required substantially longer sampling times and struggled to generate stably converged free-energy profiles due to the slow dynamics of cooperative pore water diffusion even in the barrierless limit. Using umbrella sampling, well-converged free-energy profiles can be readily generated for the wild-type BK channels as well as three mutants with pore-lining mutations experimentally known to dramatically perturb the channel gating voltage. The results show that the free energy of pore hydration faithfully reports the gating voltage of the channel, providing further support for hydrophobic gating in BK channels. Free-energy analysis of pore hydration should provide a powerful approach for quantitative studies of how protein sequence, structure, solution conditions, and/or drug binding may modulate hydrophobic gating in ion channels.

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Conflict of interest statement

Declaration of interests The authors declare no competing interests.

Figures

None
Graphical abstract
Figure 1
Figure 1
BK channel structure and key features. (A) Overall conformation in the Ca2+-free and presumably deactivated state. The structure was derived from PDB: 6V3G and further refined by MD simulations (see materials and methods). Voltage-sensing domain is drawn in red, the PGD in silver, and the cytosolic-tail domain in blue. (B) Two opposing PGD S6 helices and the selectivity filter (residues 273–330) in both the closed (silver) and open (periwinkle) conformations. A key pore-lining residue A316 is highlighted in red spheres. Potassium ions in the selectivity filter are drawn as gold spheres. To see this figure in color, go online.
Figure 2
Figure 2
Definition of pore water for BK channels. Two facing PGDs are drawn in silver cartoon from residues 230 to 330. The spherical counting region centered on the center of mass of M314 is drawn as a transparent blue sphere with a radius of 9.2 Å, which corresponds to the distance within the switching region where the occupancy is 0.5. Pore-lining residues I312, A316, P320, and E324 are drawn with the licorice style, together with water molecules within 10 Å of the central axis. Potassium ions within the filter are shown as gold spheres. To see this figure in color, go online.
Figure 3
Figure 3
MetaD simulation of pore hydration the wild-type BK channel in the closed state. (A) Nwater as a function of simulation time during a 500 ns MetaD simulation. (B) PMFs from successive 50 ns blocks drawn as an accumulation of bias (see materials and methods). See materials and methods for details of the MetaD protocol. (C) Fluctuation of the relative free energy at Nwater = 40 with respect to the minimum (near Nwater = 10) as a function of the simulation length. To see this figure in color, go online.
Figure 4
Figure 4
US for calculating the pore hydration free-energy profile of the wild-type BK channel in the closed state. (A) Nwater as a function of time during 10 ns US for all windows. The additional 10 ns of sampling in windows 15 and 20 are not shown. (B) Raw (biased) histograms of Nwater for all windows. (C) 1D PMF as a function of Nwater calculated using WHAM. Representative snapshots are shown for three selected hydration states. The S6 helix and filter region of two opposing subunits are drawn in silver. Potassium ions in the filter are drawn in gold, and pore waters are drawn as bonds with oxygen in red and hydrogen in white. Residue A316 are highlighted in cyan. Error bars shown are difference between results derived from the first and second halves of the production sampling divided by squared root of 2. To see this figure in color, go online.
Figure 5
Figure 5
Pore hydration PMF for wild-type and three mutant BK channels in both open (A) and closed (B) states. Representative snapshots are shown for the state of free energy minimum for each of the mutant channel. The S6 helix and filter region of two opposing subunits are shown in silver cartoon, potassium ions in the filter in gold spheres, and pore waters as bonds with oxygen in red and hydrogen in white. Side chains of residues at position 316 are drawn in blue, red, and green for A316I, A316S, and A316D, respectively. Error bars shown are difference between results derived from the first and second halves of the production sampling divided by squared root of 2. To see this figure in color, go online.
Figure 6
Figure 6
2D free-energy surface of pore hydration and potassium permeation. The surface was derived from US simulations as a function of Nwater and the z-position of K+ relative to the selectivity filter. Black contour lines and color bar ticks are drawn at the same energy levels and are drawn every 2 kcal/mol. 1D PMFs along Nwater and K+ positions are shown along the top and left sides, respectively. Convergence analysis is provided in Figure S7. To see this figure in color, go online.
Figure 7
Figure 7
Correlation of BK pore hydration free energy and shift in gating voltage. The experimental ΔV1/2 values were taken from Chen et al. (35). The dry and wet states are taken at Nwater = 8 and 45, respectively. The markers for A316D are an empty circle and dashed error bars to denote its approximate nature (see text). The dashed line indicates a linear fit. To see this figure in color, go online.
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