Modeling P-loops domain of sodium channel: homology with potassium channels and interaction with ligands

Abstract

A large body of experimental data on Na+ channels is available, but the interpretation of these data in structural terms is difficult in the absence of a high-resolution structure. Essentially different electrophysiological and pharmacological properties of Na+ and K+ channels and poor identity of their sequences obstruct homology modeling of Na+ channels. In this work, we built the P-loops model of the Na+ channel, in which the pore helices are arranged exactly as in the MthK bacterial K+ channel. The conformation of the selectivity-filter region, which includes residues in positions -2 through +4 from the DEKA locus, was shaped around rigid molecules of saxitoxin and tetrodotoxin that are known to form multiple contacts with this region. Intensive Monte Carlo minimization that started from the MthK-like conformation produced practically identical saxitoxin- and tetrodotoxin-based models. The latter was tested to explain a wide range of experimental data that were not used at the model building stage. The docking of tetrodotoxin analogs unambiguously predicted their optimal orientation and the interaction energy that correlates with the experimental activity. The docking of mu-conotoxin produced a binding model consistent with experimentally known toxin-channel contacts. Monte Carlo-minimized energy profiles of tetramethylammonium pulled through the selectivity-filter region explain the paradoxical experimental data that this organic cation permeates via the DEAA but not the AAAA mutant of the DEKA locus. The model is also consistent with earlier proposed concepts on the Na+ channel selectivity as well as Ca2+ selectivity of the EEEE mutant of the DEKA locus. Thus, the model integrates available experimental data on the Na+ channel P-loops domain, and suggests that it is more similar to K+ channels than was believed before.

FIGURE 1

Ligand-receptor energy obtained from the MC-minimized energy profiles of TTX and STX rotated in the channel. The zero angles correspond to the energetically optimal orientations shown in Fig. 4. Positive values correspond to the anticlockwise rotation when viewed from the extracellular side. The unimodal character of the energy profiles indicates unambiguously the preferable orientation of the toxins in the channel. The minima are 40–60° in width, indicating a certain flexibility of the complexes, which is due to the long side chains of the protein that follow the rotated ligand.

FIGURE 2

The model of the selectivity-filter region. (A) The top view of the superposition of models shaped around TTX (black) and STX (gray). Both models are practically identical in terms of backbone folding and side-chain geometry in the toxin binding sites. Sticks represent residues in the DEKA locus, Tyr⁴⁰¹, and the outer ring of acidic residues (Glu⁴⁰³, Glu⁷⁵⁸, and Asp¹⁵³²). (B) The side view showing the arrangement of large residues around Gly¹⁵³⁰. For clarity, only two pore helices are shown as ribbons. Tyr⁴⁰¹, the residue important for TTX binding (Backx et al., 1992), forms an H-bond with Glu⁴⁰³ and exposes the aromatic ring to the pore where it can interact with toxins.

FIGURE 3

The comparison of the Na⁺ channel model with the MthK template. (A) Deviations of α-carbons from the template (averaged over four repeats). (B) The α-carbon tracings of MthK (black) and Na⁺ channel (gray). Only repeats I and III are shown for clarity.

FIGURE 4

The binding of TTX (A–C) and STX (D–F) in the Na⁺ channel model. (A and D) Specific contacts of TTX and STX with residues in the selectivity-filter region predicted in our models. (B and E) Extracellular views of the complexes. P-loops are shown as ribbons and the backbones in the selectivity-filter region as α-carbon tracings. (C and F) Side views of the complexes. Ligand-receptor H-bonds are shown.

FIGURE 5

The binding of TTX derivatives. (A) Chemical structures. (B) Ligand-receptor energy obtained from the profiles of MC-minimized energy of the ligands rotated in the selectivity filter. The profiles are normalized to have the lowest minimum at zero angle. Each profile has a single energy minimum that predicts the optimal orientation of the molecule.

FIGURE 6

The binding of μ-conotoxin to the Na⁺ channel model. (A and B) Top and side views at the complex. The toxin backbone is dark-shaded; pore helices of the channel are shown as white ribbons and backbones in the selectivity-filter region as α-carbon tracings. The side chains of specifically interacting residues are shown as sticks. The toxin residues are thick and the channel residues are thin.

FIGURE 7

The ligand-receptor energy obtained from the profiles of MC-minimized energy of TMA pulled through the channel mutants in which the DEKA locus is replaced by the AAAA and DEAA loci. The zero position corresponds to the level of α-carbons in the DEKA locus. Negative coordinates correspond to the extracellular direction. The profile of TMA in the AAAA mutant shows two deep minima. The first one corresponds to the electrostatic interaction of TMA with the external ring of acidic residues. The second minimum is close to the focus of pore helices' axes. Two negatively charged residues in the DEAA mutant eliminate the barrier between the minima and enable the TMA permeation.

FIGURE 8

A water molecule in the selectivity of the Na⁺ channel. Specific interactions in the DEKA locus with the water molecule agree with the model of Lipkind and Fozzard (2000).

FIGURE 9

The Na⁺ channel P-loop region model. The pore helixes are shown as ribbons. The Roman numerals label repeats. The space-filled residues in the DEKA locus are red. The space-filled residues in the downstream positions 1, 2, 3, and 4 from the DEKA locus are colored magenta, blue, cyan, and pink, respectively. (A) Extracellular view; (B) intracellular view; (C and D) side views with two nonadjacent repeats.

FIGURE 10

Top (A) and side (B) views of the KcsA crystallographic structure with the access pathway to the pore of P-loop channels highlighted. The solid ribbons in B represent the inner helices in repeats III and IV and rods show P-loops. Numbers at A denote space-filled KcsA residues in positions where mutations in Na⁺ and Ca²⁺ channels affect the action of extracellularly applied ligands. (1) Thr^C75 and Thr^D75 correspond to the Na⁺ channel residues Phe¹²³⁶Cys and Thr¹⁵²⁸Cys that are accessible for extracellular reagents (Yamagishi et al., 1997). (2) Val^D93 and Met^D96 correspond to the Na⁺ channel residues Cys¹⁵⁷² and Ile¹⁵⁷⁵ that control external access of permanently charged analogs of local anesthetics (Qu et al., 1995; Sunami et al., 2001). (3) Val^C70 and Ala^C73 correspond to the Ca²⁺ channel Phe¹¹¹² and Ser¹¹¹⁵ that control the access of dihydropyridine to the receptor (Yamaguchi et al., 2003).