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. 2006 May 15;90(10):3555-69.
doi: 10.1529/biophysj.105.080432. Epub 2006 Feb 24.

Voltage-dependent hydration and conduction properties of the hydrophobic pore of the mechanosensitive channel of small conductance

Steven A Spronk  1 , Donald E Elmore, Dennis A Dougherty
Affiliations

Voltage-dependent hydration and conduction properties of the hydrophobic pore of the mechanosensitive channel of small conductance

Steven A Spronk et al. Biophys J. .

Abstract

A detailed picture of water and ion properties in small pores is important for understanding the behavior of biological ion channels. Several recent modeling studies have shown that small, hydrophobic pores exclude water and ions even if they are physically large enough to accommodate them, a mechanism called hydrophobic gating. This mechanism has been implicated in the gating of several channels, including the mechanosensitive channel of small conductance (MscS). Although the pore in the crystal structure of MscS is wide and was initially hypothesized to be open, it is lined by hydrophobic residues and may represent a nonconducting state. Molecular dynamics simulations were performed on MscS to determine whether or not the structure can conduct ions. Unlike previous simulations of hydrophobic nanopores, electric fields were applied to this system to model the transmembrane potential, which proved to be important. Although simulations without a potential resulted in a dehydrated, occluded pore, the application of a potential increased the hydration of the pore and resulted in current flow through the channel. The calculated channel conductance was in good agreement with experiment. Therefore, it is likely that the MscS crystal structure is closer to a conducting than a nonconducting state.

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Figures

FIGURE 1
FIGURE 1
(A) Side view of the MscS homoheptamer, colored by subunit. (B) An individual subunit with domains labeled. (Yellow) Side chains of pore-lining L105 and L109; (red sphere) Cα of V91, the upper boundary of the periplasmic vestibule; (purple sphere) Cα of G140, the lower boundary of the cytoplasmic vestibule in the truncated MscS model. The red and purple boxes mark the approximate regions of the periplasmic and cytoplasmic vestibules, respectively. The arrow marks the end of the middle-β domain, the terminus of the simulated protein. (C) The periodic box of the MD simulation system. White, protein; green, phospholipid chains; yellow, phospholipid headgroups; blue, water molecules; red, ions.
FIGURE 2
FIGURE 2
Schematic of all wild-type simulations, indicating their salt content, initial hydration state (Rh, Uh = hydrated, R, U, M, H = empty), presence of restraints (R = restrained, U, M, H = unrestrained), applied electric field (in mV/nm), and start and end times (in nanoseconds).
FIGURE 3
FIGURE 3
Pore water occupancy of Rh (A) and R (B) simulations with various electric fields. For clarity, R+50, R−50, and R−100 are not included in panel B. They have water behavior very similar to R+100 (black). (C) Probability distributions of water occupancy in R and Rh for various electric fields.
FIGURE 4
FIGURE 4
Pore water occupancy in U (A) and Uh (B) simulations. (C–E) Snapshots of the pore viewed from the periplasm. C shows the crystal structure, and D and E show frames from the end of U0 and U+100, respectively. The protein is colored by subunit, except L105 and L109, which are in yellow spacefilling.
FIGURE 5
FIGURE 5
(A) Net alignment of water dipoles as a function of position within the simulation system for various electric fields. To minimize the influence of water molecules that have a z-value corresponding to the pore region but that are in fact embedded in the membrane, only water molecules that occupied the pore at some point in the simulation are considered. From darkest to lightest (top to bottom in the pore region), the lines represent fields of +100, +50, +20, 0, −50, and −100 mV/nm. Important regions of the simulation system are marked. Light gray vertical stripe, pore region; black dashed vertical stripes, the limits of the bilayer; gray dashed vertical stripes, the limits of the protein. (B) Probability distribution of interaction energies of water molecules in the bulk and pore regions under the application of various electric fields. Dipole-field interactions are included in the energies. All data are from R and Rh simulations.
FIGURE 6
FIGURE 6
Total charge flow by conduction (A) and diffusion (B) as a function of time for unrestrained simulations in low, medium, and high salt. Fields of both +100 (positive charge movements) and −100 mV/nm (negative charge movements) are represented. Steady-state times (in nanoseconds) are as follows: (A) U+100: 3.285–9.965; M+100: 3.380–4.805; H+100: 2.005–4.295; U−100: 4–8; M−100: 2–4.5; H−100: 2.840–5.790; (B) U+100: 4.140–9.955; M+100: 2.780–4.995; H+100: 2.450–4.480; U−100: 4.04–8; M−100: 0–4.5; H−100: 2.945–6.700. (C) Comparison of total conductive charge flow in U+100 and Uh−100.
FIGURE 7
FIGURE 7
Negative ion electrostatic potential profiles for the simulation system under various electric fields in the R and Rh simulations. The plots for some fields are incomplete because no chloride ions were found in the pore region in some simulations. Important regions of the simulation system are marked as in Fig. 5 A.
FIGURE 8
FIGURE 8
Pore profiles, calculated by HOLE, for the crystal structure (black), U0 (dark gray), and U+100 (light gray). The profiles represent the pore at 3 ns in each simulation. The U+50, U+20, and U−50 profiles are similar to that for U0, and the U−100 profile is similar to that for U+100. Tick marks on the y axis are 1 nm apart. The data have been reflected across the y axis to create the appearance of a channel cross section.
FIGURE 9
FIGURE 9
(A and B) Distance of each subunit from the pore center as a function of time for U0 (A) and U+100 (B). The distance represented is that from the center of mass of the seven L109 Cα atoms to the Cγ of L109 of each subunit. (C–E) Snapshots of TM3 of the E, F, and G subunits at the end of U+100. Red spheres represent the Cα of G113, and the blue sphere represents the Cα of A107-F. TM3-E is representative of a helix that remains similar to the crystal structure.
FIGURE 10
FIGURE 10
RDF of water oxygen atoms around chloride ions in the bulk and pore regions in U+100. The pore RDF is the average of the individual RDFs of the 33 chloride ions that were conducted in the steady-state regime, weighted by the length of time the ion spent in the pore.
FIGURE 11
FIGURE 11
(A) View of the charged residues in the TM domains. Only two subunits are shown for clarity. (Blue) R46; (green) R54; (red) D67; (purple) R74; (cyan) R88; (yellow) L105 and L109. (B–D) Probability distributions of pore water occupancies in unrestrained simulations of R46 mutants (B), R74 mutants (C), and other mutants (D).
FIGURE 12
FIGURE 12
Positions of the centers of mass for TM1 and TM2 averaged over the final 0.5 ns of some simulations, relative to that for U0. The positions are also corrected for fluctuations in the position of the lipid bilayer. (A) U simulations of wild-type MscS with different electric fields. (B) Mutant simulations with an electric field of +100 mV/nm.
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