Aryal P et al. (2014), A hydrophobic barrier deep within the inner por...

XB-ART-51609

Nat Commun 2014 Jul 08;5:4377. doi: 10.1038/ncomms5377.

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A hydrophobic barrier deep within the inner pore of the TWIK-1 K2P potassium channel.

Aryal P , Abd-Wahab F , Bucci G , Sansom MS , Tucker SJ .

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Recent X-ray crystal structures of the two-pore domain (K2P) family of potassium channels have revealed a unique structural architecture at the point where the cytoplasmic bundle-crossing gate is found in most other tetrameric K(+) channels. However, despite the apparently open nature of the inner pore in the TWIK-1 (K2P1/KCNK1) crystal structure, the reasons underlying its low levels of functional activity remain unclear. In this study, we use a combination of molecular dynamics simulations and functional validation to demonstrate that TWIK-1 possesses a hydrophobic barrier deep within the inner pore, and that stochastic dewetting of this hydrophobic constriction acts as a major barrier to ion conduction. These results not only provide an important insight into the mechanisms which control TWIK-1 channel activity, but also have important implications for our understanding of how ion permeation may be controlled in similar ion channels and pores.

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Species referenced: Xenopus
Genes referenced: gnl3 kcnk1

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	Figure 1. TWIK-1 cytoplasmic gate remains open.Top: pore-lining structure and surface of TWIK-1 at the start (0âns), and end (100âns) of the MD simulation. The pore-lining helices and C-helix are highlighted. The position of the membrane is shown by the phosphorus atoms of POPC highlighted as light grey spheres. The C-helix remains stable and associated with the membrane throughout the simulation (Supplementary Fig. 1). Below each snapshot of the simulation is an expanded view of the inner-pore section indicated by the line in the top panel. Distances along the z-axis of the inner pore start below the S4 binding site. The pore radius dimensions show that the cytoplasmic mouth of the inner cavity remains open throughout the simulation. The HOLE surface profiles are coloured red (r<1.2âÃ) or blue (r>1.2âÃ).
	Figure 2. Dewetting of the inner pore.(a) Snapshots of water molecules (cyan) inside the inner pore of TWIK-1 channel at different time points during the MD simulation. For orientation, the K+ ion at the S4 site is shown as a purple sphere. Dashed lines (5â10âÃ below the S4 site) indicate the location where dewetting is most prominent. The number of water molecules in this region is indicated below each snapshot. (b) Water count within this region of the inner pore sampled every 0.1âns during the simulation. (c) Average water density inside the inner pore during the simulation is shown as transparent cyan surface contoured at 0.50 of bulk water density, overlaid on a snapshot of the inner pore at 100âns. (d) Normalized probability histogram of the water count at this region for two 100-ns MD simulations. This reveals water molecules to be absent from this region of the inner pore for >50% of the duration of the simulation.
	Figure 3. Hydrophobic cuff within the TWIK-1 inner pore.(a) A longitudinal section through the centre of the TWIK-1 channel at the end of the MD simulation. Left, a section of the average water density is overlaid. The S4 K+ ion is shown as a purple sphere. Note the hydrophobic nature residues lining the inner pore. Right, leucine residues contributing to the hydrophobic cuff are labelled. (b) Bottom-up view of the inner pore. Leu146 and Leu261 which form the hydrophobic cuff are shown as grey van der Waals spheres. M2 is shown in blue and M4 in orange.
	Figure 4. Mutation of Leu146 hydrates the inner pore.Average water density from a 100-ns MD simulation of: (a) L146D and (b) L146N mutants. The heavy atoms of the mutant side chains are shown as van der Waals spheres at the end of the MD simulation (see also Fig. 2c). Dashed lines represent approximate position of the hydrophobic cuff (c) Left, representative whole-cell currents recorded from the WT TWIK-1* and L146N-mutant channels. Right, averaged currentâvoltage relationship for whole-cell currents of WT TWIK-1* (black), L146D and L146N TWIK-1* mutants (red) expressed in Xenopus oocytes.
	Figure 5. Positional restraint simulations and energetic profiles.Dewetting precedes structural changes in the inner pore during the soft positional restraint simulation (a) Water count within the hydrophobic gate region (as in Fig. 2b) during a positional restraint simulation for WT (black) and L146N (red) sampled every 0.01âns. Positional restraint with a force constant of 10âKJâmolâ1âÃâ2 were applied to the CÎ± carbons of WT and in-silico L146N mutants (b) Normalized probability histogram of the water count at the hydrophobic cuff during the positional restraint simulations. This shows that ~30% of the simulation time, there is no water in the hydrophobic region (ÎÎ¼ nH2O (L146NâWT)=10.37). (c) and (d) Cross-section of inner pore of the starting structures for probability mass function calculations where nH2O(WT)=0 and nH2O(L146N)=12. (e) The starting PMF structures, WT structure (black) and L146N structure (red) have a comparable radius profile. (f) Potential mean force profile for a K+ ion translated along the z-axis of the inner pore of WT channel (black) and L146N-mutant channel (red). The shaded red area represents the additional free energy barrier experienced in the WT channel relative to the L146N-mutant within and above the hydrophobic cuff.
	Figure 6. L146N mutation directly alters conduction pathway.(a) Typical currents recorded from giant excised patches expressing WT TWIK-1. Almost no current can be recorded with K+ as the permeant ion in the intracellular bath solution. But replacing K+ in the bath by Rb+ produced large currents at depolarizing voltages. These TWIK-1 Rb+ currents are sensitive to block by quinine (not shown), but are not inhibited by 50-Î¼M THexA. Similar recordings of L146N-mutant channels exhibit larger currents with both K+ and Rb+ as the permeant ions, but the L146N Rb+ currents are almost completely inhibited by 50-Î¼M THexA applied intracellularly. (b) Voltage dependence of Rb+ currents for both wild-type TWIK-1* and L146N normalized to activation at +120âmV. Note that L146N currents activate over a lower voltage range. (c) Percentage remaining current for WT TWIK-1* and L146N-mutant channels after block by 50-Î¼M and 1-Î¼M THexA; L146N channels exhibit a markedly enhanced sensitivity to THexA.
	Figure 7. Representative current traces of TWIK-1* channels with different hydrophilic or hydrophobic substitutions at Leu146.(a) Currents were recorded from a series of voltage steps between â120 and +40âmV with a holding potential of â80âmV. (b) Mean currents at 0âmV for a range of different of hydrophobic (grey) and hydrophilic (red) substitutions at the Leu146 position (WT TWIK-1* is shown in black).
	Figure 8. Hydrophilic substitutions at Leu261 also hydrate the inner pore.(a) Average water density of the L261N-mutant channel structure. The heavy atoms of the mutant side chains at the end of the MD simulation are shown as van der Waals spheres (see also Fig. 2c). Dashed lines represent approximate position of the hydrophobic cuff. (b) Representative whole-cell currents recorded from L261N and L146N-/L261N-mutant TWIK-1* channels. (c) Mean whole-cell currents recorded at 0âmV for a series of hydrophobic (grey) and hydrophilic (red) substitutions at the Leu261 position. WT TWIK-1* is indicated in black. (d) Combining mutations at both Leu146 and Leu261 markedly enhances whole-cell currents for hydrophilic, but not hydrophobic substitutions.

References [+] :

Anishkin, Hydration properties of mechanosensitive channel pores define the energetics of gating. 2010, Pubmed