Mainstream- Fringe- and Patho- Physiology of Voltage Dependent Na⁺ (Na_V) Channels

Voltage gated Na⁺ (Na_V) channels mediate action potential genesis and propagation in most excitable cells. Other roles played by Na_V channels in both excitable but also in non-excitable cells highlight their role as Na⁺ influx mediators in response to stimuli other than strong and brief depolarization of the cell’s membrane.

Introduction

The lipid membrane that surrounds the cell is impermeable to charged ions that are unequally distributed across it and this unequal distribution of charged particles generates polarization of the membrane potential (more negative inside). Ion channels are proteins that span the membrane and form channels through which these ions can pass and generate ionic electrical currents. Most channels exist in either an open or shut conformation and the transition between these two main conformations is highly controlled. Voltage sensitive channels respond to changes in membrane potential by transition to a conducting conformation¹.

Voltage dependent Na⁺ (Na_V) channels are sensitive molecular devices that respond to membrane depolarization with a transient Na⁺ influx². This activity switches on the action potential (AP), the principal neuronal and muscular electrical signal¹. This review focuses on Na_V channels and their role in other cellular process ranging from neuronal modulation to cancer.

Structure, Nomenclature and Distribution of Na_V Channels

Na_V channels belong to the larger protein superfamily of voltage dependent channels that also include the K_V and Ca_V channels³. The archetypical voltage sensing K_V channel is formed by tetramerization of similar pore forming subunits and is also true for bacterial Na⁺ channels⁴. However, in higher organisms, Na_V genes encode a protein with four similar domains, each corresponding to a single subunit in the K_V channel tetramer³. Nine related genes encoding Na_V channels have been identified in mammals, all forming one protein family (See Table)⁵. In addition, a structurally similar gene corresponding to a Na⁺ activated channel (Na_V) which lacks the voltage sensor element, might form a second subfamily within Na_V⁵. However this will not be discussed in this review.

Na_V channels are expressed primarily in excitable tissues (nerve and muscle), with certain isoform-tissue/area specificity (See Table)^5,6. This point is nicely illustrated by the detection of the cardiac isoform Na_V1.5 in human embryonic stem cell derived cardiomyocytes⁷. However, recent findings demonstrate that neuronal Na_V1.1 and Na_V1.3 channels control the pacing of the mouse heart^8,9 and that the cardiac isoform Na_V1.5 is expressed in limbic regions of the rat brain¹⁰. In addition, several reports describe the expression of Na_V channels in non-excitable cells^11-13. This demonstrates that the tissue specificity of Na_V channels is a broad observation, but important deviations from this pattern exist.

The nine human Na_V channel genes are clustered on different chromosomes. Apparently structural, functional and pharmacological characteristics separates the channels encoded on chromosome 3 from all the others. This is manifested mainly by Tetrodotoxin (TTX) sensitivity demonstrated by all but the Na_V1.5, 1.8 and 1.9 isoforms (See Table)^5,6.

Na_V Channels as AP Initiators

Depolarization of the resting membrane potential is caused by the accumulation of positive charges on the inner interface of the plasma membrane. Thus, Na⁺ inflow results in membrane depolarization. This positively charged ion influx causes further depolarization of the membrane, which activates neighboring channels. This mechanism forms a positive feedback loop that lies at the heart of membrane excitability, as it forms the rising phase of most APs¹. AP is a regenerative voltage spike, traveling along the cell membrane, by means of ionic currents and activation of voltage dependent ion channels (K_V and K_Ca channels, co-localized with Na_V, repolarize the cells’ membrane, terminating the signal). In neurons, AP travel rapidly for long distances along axons, transmitting cellular coded information.

Thus, most of the nine human genes encoding Na_V channels participate in AP generation in neurons and muscle cells^2,5. The combination of Na_V channel isoforms expressed by an excitable cell determines parameters such as AP firing frequency, which are in the basis of neuronal coding. Indeed, different cell types express different combinations of Na_V channels (See Table) and alterations in such expression patterns occur in different pathophysiological conditions. For example, changes in the levels of functional Na_V channel expression forms the basis of changes in conduction properties of DRG neurons associated with chronic pain phenomena⁶. Recently, it was shown that the ubiquitin system participates in the regulation of Na_V1.5 channel functional membrane expression in the heart¹⁴, adding another dimension to the regulation of cardiac excitability.

Na_V Channel Influxes Near the Resting Membrane Potential and in Non-Excitable Cells

Usually Na_V channel influxes are brief and transient, generally activating within 1 ms after depolarization and terminating within 10 ms (while the stimulus is still on, a process called inactivation)⁵. Thus, AP arise from the brief activity of Na_V channels, which respond to the extreme depolarization generated by AP. Nevertheless, several mechanisms and Na_V channels may give rise to different activities, when the membrane voltage is at rest or during mild depolarization. This corresponds both to neuronal excitability and to the emerging role of Na_V channels in non-excitable tissue physiology and pathophysiology. Both of these roles are briefly discussed below.

Neuronal modulation by Na_V channels at rest and mild depolarization may arise from voltage dependent kinetic properties of channels such as inactivation¹⁵, persistence¹⁶ and resurgence¹⁷. These phenomena control the availability of Na_V channels before and after an AP has passed, thus, influencing the probability of an AP to emerge¹⁸. Na_V channel isoforms differ in the fine-tuning of their kinetic properties. Differences may arise from intrinsic properties, or from the ability to interact with other factors¹⁹. The expression patterns of the different isoforms gives rise to a large diversity of neurons with different AP firing behavior. Indeed, long lasting changes in expression patterns of Na_V channels under pathological conditions for example, is strongly correlated with changes in such coding capabilities of neurons²⁰.

Neuronal modulation may also arise from activation of channels by means other than voltage sensitivity. In mouse DRG neurons, Na_V1.9 currents were found to be up-regulated by the inflammatory agent prostaglandin E2, in a G- protein dependent manner²¹. Another important example is the activation of the TTX-resistant Na_V1.9 channel by neurotrophic factors (NT)^22,23. This was shown in hippocampal neurons, where BDNF via TrkB receptors and Na_V1.9 channels activated a long lasting Na⁺ current (about 200 ms) and a similar current was fourfold longer in neuroblastoma cells²³. In frog sympathetic neurons, NGF upregulated both TTX-sensitive and TTX–resistant Na_V currents²⁴. On the other hand, NGF and GDNF reduced the Na_V1.3 expression that accompanied DRG axotomy²⁵. These interactions may contribute to pain sensation and pathophysiology, with differential specific interactions between sets of NTs and Na_V channels⁶. Such interactions lie at the base of differential analgesic effects of different NTs, mediated by regulation of Na_V channels in the peripheral nervous system²⁵.

All these subthreshold activities may be the link and explanation for the expression of these highly voltage sensitive channels in non-excitable cells that do not fire AP.

Ion channel activity is an important factor in volume regulation and the associated motility of cells. A related mechanism is suggested for the ability to migrate, acquired by transformed glial cells, on their way to spread brain cancer²⁶. Several Na_V channels were reported to be upregulated in prostate cancer cell lines^12,27,28. Interestingly, the magnitude of such Na_V current was shown to be dependent on environmental factors that are present in the cell culture serum²⁹. The role played by Na_V channels, is suggested by the positive correlation between the rate of invasiveness and the level of Na_V current or expression^12,30. Most importantly, TTX, a highly specific (for Na_V, but not very selective between the six sensitive isoforms), Na_V channel blocker, reduced these cells’ invasiveness¹². A possible role for Na_V channels in volume regulation was demonstrated in Jurkat T cells, where Saxitoxin (STX) sensitive Na_V channels play a role in apoptosis related cell shrinkage and death^13,31. In addition, the Na_V auxiliary subunit β3 (SCNB3) was found to be up-regulated in cancer cell lines in a p53 dependent manner, suggesting a role for Na_V channels in induced apoptosis of cancer cells³².

These observations highlight the notion that voltage activated channels might be expressed in non-excitable cells, possibly under extreme cellular conditions.

Na_V Channelopathies

Involvement of Na_V channels in patho-physiological conditions may arise either from genetic alterations in channel activity or from changes in functional expression levels.

Mutations in coding for Na_V channel proteins cause of several hereditary neuronal³³, cardiac³⁴ and muscular³⁵ diseases. In addition, the mutation may occur de-novo (neither parent carries it) and such a mutant Na_V1.5 channel was linked to Sudden Infant Death Syndrome³⁶. In many of these mutations, the channel activity is up-regulated due to reduced inactivation, thus resulting in elevated baseline activity³³.

Altered expression patterns and level of Na_V channels in pathological states is demonstrated in Multiple Sclerosis (MS) and may contribute to specific phenotypes of this disease²⁰. In addition, it was shown using specific antibodies that co-localization of Na_V with the Na⁺-Ca²⁺ exchanger, plays a role in the increased axonal degradation seen in a mouse model of MS³⁷. Na_V channel expression is also changed at sites of axonal injury, conferring altered firing behavior to these injured neuronal branches⁶. Such a mechanism is the basis of abnormal pain phenomena as well.

Pharmacology of Na_V Channels

Na_V channels are involved in several different diseases and mediation of pain and indeed, these proteins are targets for many drugs ranging from anti-epileptic to local anesthetic agents⁵. In addition, many venomous organisms target their prey’s or attacker’s Na_V channels³⁸. In general, Na_V pharmacophores are classified according to their receptor site on the channel. Among these, several molecules block the channel, while others support increased activity, either by enhancement of activation or by slowing inactivation⁵.

TTX, STX and μ-Conotoxins modulate Na_V channels by blocking the pore from outside. TTX (See Table) and STX are largely non-selective between the different Na_V isoforms⁵, while μ-Conotoxins are more specific and may differentiate between Na_V current components^39,40. These agents are widely used to block conduction in excitable tissue in a range of experimental procedures.

Local anesthetics and related compounds, such as QX-222 and QX-314, block Na_V channels by binding to an intracellular site near the pore. These agents are capable of blocking all Na_V isoforms when applied intracellularly. However, once applied from outside, it blocks channels differentially. This is probably correlated to the ability of these agents to penetrate the cell via the open channel⁴¹. Since these compounds can exert their blocking effect only once the channel has opened, they are called open channel blockers or activity dependent blockers.

Many venoms from different animals contain peptides that are Na_V channel activators. Scorpion venoms are the largest source described so far³⁸. Both α and β scorpion toxins bind to extracellular moieties on domain IV and II of the channel to slow inactivation or enhance activation kinetics, respectively⁵. Sea anemone Na_V toxins such as Anthopleurin-C, APE 1-2⁴² or ATX II^{43, 44}, bind to the α scorpion toxin binding site and exert similar effects on inactivation⁴. Two small peptide toxins found in wasp venom, α and β-Pompilidotoxin, and promote Na_V currents by slowing the inactivation process¹⁹. Such toxins are used to mimic, or specifically up-regulate, Na_V subthreshold activities such as persistent and resurgent currents. Therefore, these Na_V channels toxin activators might be used as powerful tools in Na_V current induction at rest or in non-excitable cells.

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