Voltage Gated Sodium Channels
Michelle Wu
August 6, 2011
2011
Abstract:
The voltage gated sodium channel was first discovered in 1980. It is important
physiologically, and is a useful drug target. Voltage gated sodium channels are essential in the
nervous system and the generation of action potentials. Its structure is similar to that of most
other voltage gated ion channels: subunits arranged in such a way so that a central pore is
formed. Ions travel through the pore on electrochemical gradients. There is a lot of current
research on voltage gated sodium channels. Recently, the crystal structure for the voltage gated
sodium channel was discovered and published. Voltage gated sodium channels are quite similar
to voltage gated potassium channels, but they differ slightly as well. Voltage gated sodium
channels are a topic of great interest.[1-14]
Introduction/Background:
The voltage gated sodium ion channel regulates many physiological functions. This
makes the sodium channel an optimal drug target. About 13% of known drugs target ion
channels for primary therapeutic action. In particular, sodium channels are targets for anesthesia
and treatments for genetic diseases in the brain, skeletal muscle, and heart. The physiological
importance of the voltage gated sodium channel is highlighted by its association with numerous
pathologies. Over fifty-five channelopathies (inherited ion channel diseases that result from a
disturbance to a subunit) have been identified, involving the cardiovascular, neuronal,
neuromuscular, musculoskeletal, metabolic, and respiratory systems.[1] Some well-known
diseases associated with the voltage gated sodium channel include: epilepsy, familial hemiplegic
migraines, and familial autism. Most of these diseases are caused by mutations to genes encoding
the channel. For example, mutations to the gene SCN1A results in these channelopathies: febrile
epilepsy, Doose, Dravet, and West syndrome, familial hemiplegic migraines, familial autism,
and more. These diseases can range from mild to life-threatening. Channelopathies are generally
gain-of-function: a mutation conferring new or enhanced activity on the protein occurs. Loss-offunction mutations occur too, but rarely. Drug discovery is difficult in this area. As high
throughput molecular approaches are ineffective, most ion channel drugs are discovered using
tissue and animal based pharmacological methods.
Voltage gated sodium channels are integral to the nervous system. They are crucial to the
generation of nerve impulses. In 1952, Hodgkin and Huxley first analyzed the properties of the
ion channel. They proposed three key features: voltage dependent activation, rapid inactivation,
and selective ion conductance. Analysis of the sodium channel function occurred in the 1960’s,
and in 1980, the voltage gated sodium channel was discovered.[2] The sodium channel became
the first voltage gated ion channel to be cloned and sequenced not long after. The voltage gated
sodium channel belongs to the superfamily of ion channels. It was the first to be discovered in
that superfamily.
Sodium Channel Function: The Action Potential
The voltage gated sodium channel tends to reside in electrically excitable cells. It is,
however, expressed at low levels in non-excitable cells, though its physiological role there is
unclear. In excitable cells (generally neurons, muscle and endocrine cells), the sodium channel
plays an essential role in the initiation and propagation of action potentials. During an action
potential, the electrical membrane potential of a cell will rise and then fall. Action potentials are
initiated when a stimulus causes the membrane potential to reach a threshold, which in turn
results in depolarization. There is a wide variety of stimuli. After the membrane potential reaches
a threshold, voltage gated sodium channels open, allowing an influx of positively charged
sodium ions into the cell and further depolarizing it. Repolarization to resting potential occurs
when the sodium channels are inactivated and potassium channels are activated. The sodium
channels’ inactivated state temporarily prevents them from reopening even though the cell is still
depolarized. Once repolarization occurs, the sodium channels revert to a closed state, ready to
open at the next action potential. The action potential takes only a couple milliseconds, and is an
all-or-nothing action. (See Figure 1). In neurons, action potentials function in cell to cell
communication. In other cells, action potentials activate intracellular processes.
http://faculty.washington.edu/chudler/ap3.gif
Figure 1: Action potential diagram: shows voltage and time in relation to the opening and closing of sodium channels.
Structure of the Voltage Gated Sodium Channel
The voltage gated sodium channel is composed of a large alpha subunit, generally around
2000 amino acids long. At times, the sodium channel will associate itself with other proteins.
The alpha subunit is the core of the channel and is functional on its own. As long as the alpha
subunit is expressed, channel opening, ion selection, and rapid inactivation can all take place.
The primary sequence of the voltage gated sodium channel is the alpha subunit folded into four
domains (I-IV). These domains are non-identical but similar, and each contains six alpha-helical
transmembrane regions (S1-S6). S1-S4 is the voltage sensing domain, and S4 is the voltage
sensor. S4 contains several positively charged residues and two hydrophobic residues responsible
for voltage sensing. The narrow ion-selective filter is formed by reentrant loops between the S5
and S6 helices. (See Figure 2). Large intracellular loops link the four domains.[3] The voltage
gated sodium channel is a tetramer, and therefore rotationally symmetric. The domains are
arranged circumferentially around the central pore, so the intracellular part of each domain has a
voltage sensing region and a pore forming region.[4] The voltage gated sodium channel also
contains one or more beta subunits. Beta subunits are multi-functional, regulating channel gating
and modulating the level of channel expression. They are also cell adhesion molecules. Beta
subunits are viable future therapeutic targets because of their ability to regulate alpha subunits.
Four beta subunits have been identified.
http://genomebiology.com/content/figures/gb-2003-4-3-207-1-l.jpg, http://scienceblogs.com/afarensis/upload/2006/03/channel.jpg
Figure 2: a) Four domains of a voltage gated sodium channel. Pore is labeled between S5 and S6. The S4 helices are colored yellow. Two beta
subunits are also shown. The reentrant loop in each domain marks a selectivity filter. The four domains are not identical. b) 3D structure of alpha
subunit. c) Schematic representation of one domain of the voltage gated sodium channel. S4 is yellow, S5 and S6 are green. Figure 2: 3D
computer image of alpha subunit.
The central pore opens when all four voltage sensors activate, according to kinetic
models. It seems to dilate when the voltage sensor domains and S4 and S5 linkers pivot together
around the base of the pore, as per comparisons to open pore potassium channel structures. The
voltage sensing domain and S4-S5 linker pull the S5 and S6 helices outward to open the pore.
Neighboring subunits are forced to move likewise because of tight structural coupling.[6]
The Crystal Structure of the Sodium Channel
The crystal structure of the voltage gated sodium channel was published July 10, 2011 by
Payandeh, Scheuer, Zheng, and Catterall. The four reported their findings on “the crystal
structure of a voltage-gated Na+ channel from Arcobacter butzleri (NavAb) captured in a closed
pore conformation with four activated voltage sensors at 2.7 Å resolution”.[5] (See Figure 4).
They found that the NavAb (type of bacteria) selectivity filter is short and water filled, with four
acidic side chains around the ion conduction pathway’s narrowest region. (The ion conduction
pathway is highly electronegative). A reentrant loop between the S5 and S6 helices forms the
narrow ion-selective filter.[6] Partial dehydration confers sodium selectivity.
The discovery of the crystal structure revealed a basis for selectivity and high
conductance. The pore of the channel consists of an outer vestibule, central cavity, a selectivity
filter, and an intracellular activation gate.[5] The central cavity is large enough to allow a sodium
ion and its first hydration shell through, and also provides a hydrophobic surface for the atoms to
diffuse over. Cations in the central cavity are stabilized by pore helices through helical-dipole
interactions. The selectivity filter, also in the pore, is one of the narrowest regions around the
extracellular side of the membrane. It contains a high-field-strength anionic site. Studies show
that Glutamic acid (Glu) side chains are key elements in determining selectivity. Glu177 side
chains form a scaffold 6.5x6.5Å. These side chains are supported by a network of interactions
(Threonine175 at the end of P-helix accepts a hydrogen bond from Trytophan179 to connect to
adjacent subunits, and Glu177 side chains form hydrogen bonds with backbone amides from
Serine189 and Methionine181). More hydrogen bonds between amino acids further stabilize the
filter.
The pore radius is large enough to accommodate a partially hydrated sodium ion. A
sodium ion surrounded by four water molecules could interact with the backbone carbonyls of
amino acids. Once through the filter, full rehydration would occur via interactions with the
water-filled sites formed by Leucine176 and Threonine175 backbone carbonyls. Free diffusion
then enables the sodium ion to enter the central cavity, activation gate, and finally the cytoplasm.
Sodium selectivity is important in the channel’s function, nerve impulses.[5]
Glu177 side chains are also blocking sites for toxins such as tetrodotoxins and
saxotoxins. On the intracellular side, local anesthetics, antiarrhythmic and antiepileptic drugs
could block the channel. Drug molecules easily fit into the central cavity of the pore. Pore
opening is required for large or hydrophilic drugs to access the binding sites because of the
presence of a seal on the intracellular activation gate.
Details about the voltage sensing domain were also discovered. S3 has a dynamic
connection with S4 (the voltage sensor). The S3-S4 loop is not rigid, which allows it to
accommodate the S4 region’s large movements. The S2-S3 loop
http://www.pdb.org/pdb/images/3rvy_bio_r_500.jpg
Figure 4: Crystal structure of voltage gated sodium channel as discovered by Payandeh, Scheuer, Zheng, and Catterall.
Nomenclature
Sodium channel genes have been found in many organisms, such as flies, leeches, squids,
jellyfish, and mammalian and non-mammalian vertebrates. There are a variety of sodium
channels, discovered by electrophysiological recording, biochemical purification, and cloning.
Originally, the various sodium channels were named in inconsistent ways, but eventually a
standard nomenclature for voltage gated sodium channels was developed to avoid confusion. The
nomenclature for sodium channels is based off that of the potassium channel; it is a numerical
system that defines subfamilies and subtypes based on similarities in amino acid sequences. Take
Nav1.1, for example. The first part, Na, is the ion’s chemical symbol. The next part, in this case,
v, is the principal physiological regulator (voltage). The 1 represents the gene subfamily, and the
number following the decimal indicates the order in which each gene was identified. Nine
mammalian sodium isoform channels have been identified so far. There are 3 isoforms in the
central nervous system (Nav1.1-1.3), 3 in the peripheral nervous system (Nav1.7-1.9), 1 isoform
in both the central and peripheral nervous systems (Nav1.6), 1 that resides exclusively in skeletal
muscle (Nav1.4), and 1 isoform mainly found in the heart, but also in the central nervous system
(Nav1.5).[7]
Engineering Sodium Channels
There is a lot of current research on voltage gated sodium channels. As mentioned earlier,
the crystal structure of the voltage gated sodium channel was recently discovered. New ion
channel drugs are also being researched. However, this is difficult. High-throughput molecular
approaches as well as traditional electrophysiology are highly inefficient. Thus, automated
electrophysiology instruments were discovered. These instruments are precise and use
automation to increase throughput. Electro-optical technologies are also being developed. The
electrical stimulation voltage ion probe reader (E-VIPR) measures voltage gated sodium channel
activity using extracellular electrical field stimulation and voltage sensitive fluorescent probes.[8]
Many scientists are also using cloning methods. Scientists hope to gain insight as to why the
voltage gated sodium channels only allow sodium ions in. They also hope to find a better pain
medication with fewer side effects and improved treatment. NavAb is useful in pharmacology:
Glu177 side chains provide a blocking site in sodium channels. Receptor sites on the S6
segments are also utilized by local anesthetics, antiarrhythmic, and antiepileptic drugs. Other
blockers are toxins such as tetrodotoxin and saxitoxin. At least 6 receptor sites have been
identified. They are formed by amino acid residues from P-loops. Drug molecules can easily fit
in the central cavity of the pore. There is, however, a seal at the intracellular activation gate.
Thus, pore opening is required for large or hydrophilic drugs to enter and proceed to the S6
receptor sites.
Comparisons
Voltage gated sodium channels have a similar structure to potassium channels and are
therefore thought to have evolved from the potassium channel by gene duplication.[6] For
example, the voltage sensing domains of the sodium and potassium channels are similar. There is
an equivalent displacement when the pore opens. During pore opening, the voltage sensing
domain and S4-S5 linkers move as a unit. Then, a molecular hinge at the base of S5 mediates
closed to open pore transitions. Lastly, tight structural coupling is maintained, and the S5 and S6
helices are pulled away, opening the pore.[5] The S4 on both potassium and sodium channels also
contain positively charged residues for voltage sensing. Another similarity between the two
channels is that when their activated voltage sensing domains are overlaid, the S4 and S5 linker
superimpose perfectly.[5]
However, a difference is that selectivity is achieved differently in the sodium channel. In
potassium channels, main chain carbonyls create the selectivity filter. It is very narrow and
conducts mostly dehydrated potassium ions. In the sodium channel, amino acid chains form the
selectivity filter. The four P-loops of the sodium channel are spread out, leaving a short
selectivity region. In the selectivity filter, cation selection is with positively and negatively
charged residues from the four domains. Four key residues have been identified through cloning:
aspartate, glutamate, lysine, and alanine (DEKA). There is one amino acid from each of the Ploops from each domain. The selectivity filter in the potassium channel is also much narrower.
While sodium channels conduct partially hydrated sodium ions, potassium channels conduct
almost fully dehydrated potassium ions through backbone interactions with backbone carbonyls
in a long narrow pore.
Another difference between sodium and potassium channels is that their amino acid
sequences are not highly analogous.
Proposed Modification
Purpose: To find out more about how the selectivity filter works.
An unsolved mystery of the voltage gated sodium channel is why the channel selects
sodium so efficiently. We know that Glu177 side chains are important in ion selectivity. They act
as water molecule acceptors for the two of the water molecules surrounding a sodium ion as the
ion enters the pore. So what would happen if the channel was modified, and the Glu177 side
chains removed? A simple procedure for this modification could be done by computer
simulation. If the selectivity filter was isolated from the voltage gated sodium channel, and a
sodium ion was placed near it, we could minimize energy and calculate the distances of where
the sodium ion ended up. Then the procedure could be repeated, without the Glu177 side chains.
After minimizing the energy and calculating distances, data could be compared.
If the Glu177 side chains were removed, most likely the sodium channel would no longer
select sodium very efficiently. But how would this affect the rest of the channel structure? Would
the selectivity filter still be able to function? What other consequences could there be?
Conclusion
The voltage gated sodium channel is present in many organisms. It plays a crucial role in
action potentials, and generally resides in excitable cells. Nine isoforms of the voltage gated
sodium channel have been discovered Channelopathies are diseases associated with mutations to
the channel. The crystal structure of the voltage gated sodium channel, discovered recently, led
to new ideas, such as how a sodium ion travels through the selectivity filter and pore. Though
the voltage gated sodium channel is thought to have evolved from the voltage gated potassium
channel, they still have their differences. There is still ongoing research on this topic.
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Acknowledgements
Thanks to Professor Allen and Slava for their advice and help. My gratitude also goes to
Eric and Ming for their patience, explanations, tips, and support. Thanks to David van Muyden
as well for his constant support.