Three-dimensional Modelling of the Voltage-gated Sodium Ion Channel from Anopheles gambiae Reveals Spatial Clustering of Evolutionarily Conserved Acidic Residues at the Extracellular Sites

Abstract

Background: The eukaryotic voltage-gated sodium channel(e-Nav) is a large asymmetric transmembrane protein with important functions concerning neurological function. No structure has been resolved at high resolution for this protein.

Methods: A homology model of the transmembrane and extracellular regions of an Anopheles gambiae para-like channel with emphasis on the pore entrance has been constructed, based upon the templates provided by a prokaryotic sodium channel and a potassium two-pore channel. The latter provides a template for the extracellular regions, which are located above the entrance to the pore, which is likely to open at a side of a dome formed by these loops.

Results: A model created with this arrangement shows a structure similar to low-resolution cryoelectron microscope images of a related structure. The pore entrance also shows favorable electrostatic interface.

Conclusion: Residues responsible for the negative charge around the pore have been traced in phylogeny to highlight their importance. This model is intended for the study of pore-blocking toxins.

Keywords: Eukaryotic voltage gated sodium channel; anopheles.; extracellular interface; homology model; pore blocker; toxin; transmembrane.

Fig. (1)

Structure of Nav channel. The NT (N-terminal), I1, I2 I3 and CT (C-terminal) regions are represented as bins because their secondary structure is unknown and irrelevant to this study. The structure of the CT region however has been determined for human Nav channels, and is shown to have a calcium binding function, interacting with calmodulin and other factors. The circles indicate position of ion near the filter loop, which contribute to the DEKA tetrad.

Fig. (2)

Alignment of the D1 (domain 1) p-loop region and some flanking transmembrane regions. The Anopheles sequence is represented by A5I843_ANOGA and Drosophila by SCNA_DROME. The alignment is colored by sequence identity. The SecStruct annotation gives the secondary structure used in the modelling, which takes into account the conserved regions. Human SCNs have longer lengths, with SCN4A having the longest, despite clustering close to SCNA_DROME.

Fig. (3)

Alignment of the D3 p-loop. It shows the conserved cysteines, and highlights important acidic residues.

Fig. (4)

Alignment of the D4 p-loop. This loop forms the entrance to the pore in the model and has conserved DD or ND motif. Asparagine (N) residues serve as attachment points for glycans like Sialic Acid which are negatively charged and enhance the functioning of the channel.

Fig. (5)

Proposed secondary structure configuration of the extracellular region, fitted to the template provided by the human two-pore potassium channel structure pdb:3UKM. The proposed structure takes into account the length of D1 and D3 p-loops and their conservation in alignment. At least 4 cysteine residues are present, with the KC sequence common for D1 and D3 p-loops at around the same place, implying symmetry. Sequence-based structure prediction does not predict such a structure, due to absence of structural representatives in known structures. The D2 p-loop provides the negative electrostatics necessary at the pore by the presence of aspartates, with D1 and D3 providing additional aspartate and glutamates. Attachment points for N-linked glycans are also present around the pore entrance.

Fig. (6)

The D1 p-loop region alignment used for generating the model using MODELLER. The 3UKM structure was included twice. One inclusion aligned the S5, S6 and filter residues with the corresponding sodium channel 4MS2 (with calcium ions renamed as BLK residues). Helix h1 and h2 aligned with this inclusion. The second 3UKM inclusion aligned only helix h4, with the rest of the sequence unaligned. Both inclusions of 3UKM were structurally aligned and essentially the same structure file. The filter residue of D1, which is the D of the DEKA tetrad, is marked in alignment at extreme right.

Fig. (7)

Configuration of the modelled extracellular regions with lower areas clipped. The ions which were included as block residues are seen as spheres in center. This is comparable to the schematic in Fig. 5. The domains are labelled D1 to D4, with italic labels denoting the pore subdomain and bold normal text denoting the voltage sensing subdomain. The helix positions are labeled h1 to h4.

Fig. (8)

Electrostatic surface map of the pore entrance. The contributing residues labelled E.218 is on D1 p-loop, E762 is on D3 p-loop, D1057 and D1058 are on D4 p-loop and D1092 is on D4 q-loop. The D4 p-loop DD or ND motif is conserved in Nav channels. Some ASN (N) residues present serve as attachment points for N-linked glycans such as sialic acid which contribute further to the negative charge in the entrance of the sodium channel and enhance its function.

Fig. (9)

The ribbon view of structure shown in Fig. (7). The acidic residues surrounding the mouth of the pore are visible.