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The voltage gated sodium channels (NaV) are responsible for triggering and propagation of action potentials in nerve, and muscle cells as well as the cells that transfer the signals from nervous to endocrine system (neuroendocrine cells). With NaV closed, the resting potential of a typical nerve cell of about –60 mV is sustained. The action potential is initiated with opening of NaV channels which results in a rapid influx of Na+ ions into the cell. This further causes a fast increase in membrane potential to about +30 mV that is carried along the excitable cell membranes. [1] Improper functioning of NaV has been linked to many disorders such as epilepsy, arrhythmia, migraines, and chronic pain.
Like other channels, NaV has a very high selectivity for its substrate, Na+. For example, its permeability for Na+ is 12 times greater than for the chemically very similar K+. [2]
The fully functioning NaV channels (shown here as cartoon model) consist of a larger alpha subunit (purple) and a smaller beta subunit (yellow). The structure on the right is that of NaV1.4 α subunit in complex with β1 subunit from human skeletal muscle. The nomenclature of mammalian α subunits follows simple numbering scheme NaV1.x where x = 1-9. Number "1" indicates only one recognized gene subfamily responsible for expression of NaV. The x value designates specific isoform with numbers 1-9 assigned in an approximate order of discovery. The nine known isoforms have more than 50% overlap in the primary structure. [3]
While the α subunit alone is sufficient for function, the β subunit tunes the kinetics of Na+ influx, voltage dependence and plays a role in channel localization (but see further below as well). There are four subtypes of β subunits: β1-β4. Some NaV α subunits can form complexes with any of the four β subunits; for example, NaV1.1, found in hart muscle cells. Others show a preference: NaV1.6, located in cerebellum, combines with either β1 or β2 only. [3]
Figure 1. The approximate dimensions of NaV1.4/β1 complex (click on the thumbnails).
The primary structure of both subunits is dominated by hydrophobic amino acids (white colored areas). This allows for the whole structure to nest inside the hydrophobic phospholipid bilayer of the cell membrane. The polar amino acids (red for negative and blue for positive amino acid residues) are found mostly in the areas exposed to the aqueous environment of intracellular space.
To see the potential surface map of the NaV1.4/β1 complex click on the thumbnails for Figure 2 below. On the second figure, you will notice a significant build up of the negative charge in the central area, required to attract positively charged, solvated Na+. The positive charge is concentrated around the edges of the structure making sure it is anchored to negative phosphate groups of phospholipids forming the cell membrane.
Figure 2. The potential surface map of NaV1.4/β1 complex (click on the thumbnails).
The alpha subunit is composed of one long chain of amino acids of approximately 200 kDa. Its secondary structure is composed of α helices connected via loops of varying length. In this view, the structure has been rotated for 90° for a view from outside the cell.
The tertiary structure consists of four domains: domain I (DI; purple), domain II (DII, green); domain III (DIII, red) and domain IV (DIV, blue).
Each domain has six transmembrane (TM) segments, S1-S6, here shown for DI only. These are further grouped in two subdomains: voltage sensing or VSD (segments S1-S4, yellow) and pore (segments S5 and S6, red). The two subdomanis are connected via an almost horizontal alpha helix, the S4-S5 linker (green). The S5 and S6 are connected via an extracellular loop (gray) and selectivity filter (green). The loops connecting the segments are in magenta.
Although the four domains have overall secondary and tertiary structure identical, there are differences in length of individual segments; for example segment S4 in domains I and II has six while in domains III and IV has seven helical turns (see further below). These differences result in a asymmetrical structure for NaV channel.
Two helices, P1 and P2 found in each domain and situated between S5 and S6, define the pore through which sodium cations enter. Four residues, one from each domain and located between P1 and P2, are crucial for the recognition of the cation: Asp406 (DI); Glu761 (DII), Lys1244 (DIII) and Ala1536 (DIV). They form a DEKA motif, after the one-letter amino acid code (D = Asp, E = Glu, K = Lys and A = Ala), and are conserved for NaV channels.
The DEKA motif is surrounded by negatively charged glutamate and aspartate sidechains. Their deprotonated carboxyl groups stabilize sodium cation and help guide it into the cavity while repelling anions and assisting in removing weakly bonded water molecules of primary hydration sphere around Na+.
Some structural evidence suggests that the initial binding of sodium cation occurs at the DEE site composed of Glu764, Glu761 and Asp406 residues (giving DEE in one-letter code). [4]
The two glutamates are locked in position through hydrogen bonding with Arg756. The geometric organisation of DEE site, the pore size and distribution of DEKA residues jointly provide selectivity for Na+ ion. [5]
Figure 3. The initial binding site for Na+ ion (click on the thumbnails).
The exact mechanism of voltage control is not fully elucidated but involves conformational changes in the voltage sensing domains which trigger restructuring of contacts in other parts of the structure.
The conserved sequence of basic amino acids, [Lys/Arg]X2[Lys/Arg]X2[Lys/Arg]X2[Lys/Arg], found in S4 of VSD represents basis for voltage sensing. These amino acids (shown only for domain I) are protonated under physiological pH creating a highly positive area. At resting membrane potentials, the S4 is attracted to the cytoplasmic side (a negative side the cell membrane) and the NaV channel is closed. As the membrane potential becomes more positive (depolarization), the VSD moves away from cytoplasmic side and the channel opens.
This is the view along S4 helix of domain I from the extracellular side. It shows three Arg and one Lys residues responsible for voltage sensing. The S4 helices of all four domains can be seen on Figure 4 below. The figure also emphasizes the asymmetry in four domains mentioned above.
Figure 4. The S4 helices of domains I-IV (click on the thumbnail).
The NaV channels are rapidly inactivated as the potential approaches resting values. There is strong evidence, based on mutation studies, that three hydrophobic residues Ile1310, Phe1311 and Met 1312 (ball and stick) found at the end of DIII-DIV connector (dark orange cartoon) play a crucial role in the process. (Domain IV is now shown as transparent blue cartoon). They constitute a conserved IFM motif (again based on on-letter codes for amino acids). Studies indicate that mutations at Phe1311 site particularly decrease the inactivation rate. [6]
The IFM residues interact with a hydrophobic pocket found between horizontal S4-S5 linkers in domains III and IV and S5 pore helix in domain IV. The hydrophobic residues forming the pocked are shown as ball-and-stick models colored white (a default color for non-polar protein regions).
The proper positioning of IFM motif allows for interaction between amino acids of III-IV helix and those on S4-S5 helix of domain IV. These interactions are a combination of van der Waals and electrostatic forces. Mutations of relevant residues on S4-S5 helix decreased the rate of NaV channel inactivation, as well.
The beta subunit plays important role in modulating NaV channels. However, their involvement in physiological functions is more complex than this (see ref [7]).
The structure β1 subunit consists of immunoglobulin (Ig) domain (gray) and one transmembrane (TM) helix (gray-green). They are connected through a short loop (red).
The Ig domain's secondary structure is composed of β-strands connected via lengthy loops. Its tertiary structure is defined through hydrogen bonds (yellow dashed lines) and one strong Cys-Cys disulfide bond. The Ig domain allows 7beta; subunits to function as cell adhesion molecules.
The Ig domain interacts with domains I, III and IV of alpha subunit on the extracellular side. Several amino acid residues involved in these interactions are shown as ball-and-stick models on the left. Figure 5 below provide some more detail on the specific interactions.
Figure 5. Details of Ig/β1-α subunit interactions (click on the thumbnails).
The TM domain of β-subunit is in close contact with S0 and S2 segments of domain III. Several amino acids involved in the contact are highlighted on the display. More detailed view is available on Figure 6 below as well as a short video clip. This non-covalent interaction between α and β subunits is typical for β1 and β3 types; β2 and β4 engage in covalent interactions as well with their α subunits through disulfide (S–S) bonding.
Figure 6. Details of TM/β1-α subunit interactions (click on the thumbnails).
Video: Details of TM/β1-α subunit interactions.
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The NaV channels are targets of many neurotoxins. One example, shown here, is tetrodotoxin (TTX). TTX belongs in a broader group of guanidinium toxins. (For a two dimensional structure of TTX please click here.) It is found in some fish, worms, frogs, arthropods and red coralline algae. Probably the best known occurrence of this toxin is in pufferfish, considered a delicacy in the Far East cultures (Japan, Korea, China). Considered the among the most poisonous vertebrates, preparing pufferfish for human consumption requires special skill and caution. Some evidence has shown that the fish do not produce the toxin, rather the symbiotic bacteria inhabiting the animal's gut are its source. Only in some rare cases the evidence supports biosynthesis of TTX by fish species. [8]
Figure 7. Examples of pufferfish species (click on the thumbnails).
TTX isa NaV channel blocker–it completely obstructs the entry of Na+ ions to the cell by binding in the same location where Na+ ion is recognized and moved into the cell. The blockage prevents creation of action potential and, thus, inhibits the cell excitation leading to paralysis. The toxin is about 1200 times more toxic to humans than cyanide and remains without antidote. [8] Here TTX molecule is shown blocking a NaV channel of American cockroach, viewed from outside the cell, perpendicular to the channel pore.
The high toxicity of TTX comes from strong binding to NaV channels. Protonated guanidinium group and several –OH groups found in TTX structure form strong hydrogen bonds at the DEE site (stick structure).
Additional hydrogen bonds are formed with several other polar residues at the pore, enhancing the TTX-NaV interaction.
The Tyr376 (white stick model) and TTX from π-cation interaction. This seems a particularly important contact, since the NaV channels that have Cys (NaV1.5) or Ser (NaV1.8 and NaV1.9) at this position are insensitive to TTX.
This is an overview of interactions between TTX and NaV in side-view.
Figure 8. TTX bonded to NaV channel (click on the thumbnails).