FIGURE 2.

Binding of TTX and Ca²⁺ in the revised model of NaV1.4. A and B, top and side views of TTX-bound channel. Backbones in repeats I, II, III, and IV are cyan, opaque, green, and magenta, respectively. P-helices are shown as ribbons, ascending limbs as thick α-tracings, and S6s as thin α-tracings. TTX fits perfectly in the outer pore and interacts with known TTX-sensing residues. C, cation-π interaction between Y^1p51 and guanidine group of TTX (22). This interaction is achieved by only a modest deformation of our previous model (5), in which Y^1p51 faces the pore and interacts with TTX by the aromatic ring edge rather than the aromatic plane. D, superposition of α tracings of the current model, which is MC-minimized either in the presence of Na⁺ ions (yellow Na⁺ ions, magenta side chains and backbones) or TTX (green side chains and backbones). Side chains of W^1p52, W^3p52, W^4p52, and TTX-sensing outer carboxylates E^1p53, E^2p53, and D^4p53 are shown as sticks. The root mean square deviation between α carbons in these models is as small as 0.2 Å. Note that hydroxyl groups of the TTX molecule are very close to positions of Na⁺ ions. E, possible binding of a Ca²⁺ ion in the Na⁺ channel. The ion binds to acidic residues D^1p50 and E^2p50 of the DEKA locus and displaces the ammonium group of K^3p50 toward the inner pore. The aromatic side chains of Y^1p51 and W^2p51 are close enough to the Ca²⁺ ion to interact with it through water molecules in the first hydration shell. The complex likely corresponds to the blocked channel state. In the absence of another cation-binding site in the narrowest part of the outer pore, an incoming ion cannot displace the bound Ca²⁺ ion.