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Comparative Study
. 2003 Apr;84(4):2345-56.
doi: 10.1016/S0006-3495(03)75040-1.

Filter Flexibility in a Mammalian K Channel: Models and Simulations of Kir6.2 Mutants

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Free PMC article
Comparative Study

Filter Flexibility in a Mammalian K Channel: Models and Simulations of Kir6.2 Mutants

Charlotte E Capener et al. Biophys J. .
Free PMC article

Abstract

The single-channel conductance varies significantly between different members of the inward rectifier (Kir) family of potassium channels. Mutations at three sites in Kir6.2 have been shown to produce channels with reduced single-channel conductance, the largest reduction (to 40% of wild-type) being for V127T. We have used homology modeling (based on a KcsA template) combined with molecular dynamics simulations in a phosphatidycholine bilayer to explore whether changes in structural dynamics of the filter were induced by three such mutations: V127T, M137C, and G135F. Overall, 12 simulations of Kir6.2 models, corresponding to a total simulation time of 27 ns, have been performed. In these simulations we focused on distortions of the selectivity filter, and on the presence/absence of water molecules lying behind the filter, which form interactions with the filter and the remainder of the protein. Relative to the wild-type simulation, the V127T mutant showed significant distortion of the filter such that approximately 50% of the simulation time was spent in a closed conformation. While in this conformation, translocation of K(+) ions between sites S1 and S2 was blocked. The distorted filter conformation resembles that of the bacterial channel KcsA when crystallized in the presence of a low [K(+)]. This suggests filter distortion may be a possible general model for determining the conductance of K channels.

Figures

FIGURE 1
FIGURE 1
(A) Sequence alignment of the pore helix (P) and filter (F) regions for Kir2.1, Kir6.2, and KcsA. The residues for which mutant models have been constructed (see text) are boxed, as is the Y-to-F difference in the GYG motif. Numbering is relative to the Kir6.2 sequence. (B) Pore region of the Kir6.2 model (two subunits out of four are shown). The residues which have been mutated (V127, G135, and M137) are shown in ball-and-stick format. (C) The Kir6.2 channel model (all four subunits) is shown along with the approximate location of the lipid bilayer. Aromatic side chains used to judge the location of the transmembrane region are shown in space-filling format.
FIGURE 2
FIGURE 2
Initial filter configurations (two subunits only shown) for simulations WT0, WT1, and WT3. K+ ions (green spheres) are present at S1, S3, and in the cavity. Water molecules within and behind the filter are shown (in red/white) as indicated by the arrows.
FIGURE 3
FIGURE 3
Cα RMSD of the TM region (i.e., the M1 helix, the P-helix, and filter, and the M2-helix) versus time for the WT0 (solid, black), VT0 (solid, gray), GF0 (broken, gray), and MC0 (broken, black) simulations.
FIGURE 4
FIGURE 4
Final (i.e., t = 2 ns) filter configurations (only two subunits shown), the WT simulations (with 0, 1, and 3 waters per subunit behind the filter—see Table 1 and text), for the V127T mutant simulations, for the G135F mutant simulations, and for the M137C mutant simulations.
FIGURE 5
FIGURE 5
Summary of the conformations adopted by (A) residue I131 and (B) residue G132 in all simulations as a percentage of time, averaged over the period 0.5–2.0 ns of each simulation. α-Helix (cross-hatch) is defined as 0° ≥ Φ ≥ −180° and +30° ≥ Ψ ≥ −130°; β-strand (vertical stripes) is defined as 0° ≥ Φ ≥ −180° and (+180° ≥ Ψ ≥ +30° or −130° ≥ Ψ ≥ −180°); left-handed α-helix (gray) is defined as +110° ≥ Φ ≥ 0° and +120° ≥ Ψ ≥ −30°; all other regions are defined as random coil (empty). Note that these regions correspond to rectangles in the Ramachandran plot that approximate the secondary structure regions defined in e.g., Procheck (Morris et al., 1992).
FIGURE 6
FIGURE 6
Backbone torsion angles versus time: (A) I131 Ψ and (B) G132 Φ angles, for simulation VT0. The four lines in each graph correspond to the four subunits.
FIGURE 7
FIGURE 7
Comparison of the two x-ray structures of the KcsA filter region with snapshots from the WT0 and VT0 simulations. In each case just two subunits (and two K+ ions) are shown. The structures shown are for KcsA at high [K+] (PDB code 1K4C); Kir6.2 simulation WT0 at 1 ns; KcsA at low [K+] (PDB code 1K4D); and Kir6.2 simulation VT0 (i.e., the lowest conductance mutant) at 1 ns.
FIGURE 8
FIGURE 8
Pore radius profiles for the filter regions of: (A) KcsA at high [K+] (solid line) versus Kir6.2 simulation WT0 at ∼1 ns (broken line, average of profiles at 10-ps intervals from 0.95 to 1.05 ns); and (B) KcsA at low [K+] (solid line) versus Kir6.2 mutant simulation VT0 (broken line, average of profiles at 10-ps intervals from 0.95 to 1.05 ns) at ∼1 ns. The horizontal gray line represents the radius of a K+ ion. In (C) the Kir6.2 WT0 and VT0 profiles at ∼1 ns are redisplayed to facilitate direct comparison.
FIGURE 9
FIGURE 9
Trajectories, projected onto the filter (z-) axis, of K+ ions (black lines) and selected water molecules (gray lines) within the channel. The four binding sites S1–S4 are shown as thin black lines. The diagram to the right of each graph is a schematic of the filter and cavity of the channel. Results are shown for simulations: (A) WT3; (B) VT0; and (C) GF1.

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