**2. Nav1.7**

Voltage gated sodium channels are transmembrane proteins that are formed by an alpha (α) subunit composed of approximately 2000 amino acid residues, this subunit

**Figure 1.** *Voltage-gated sodium channel structure.*

is organized in four homologous domains that make up the Na<sup>+</sup> channel pore. Each domain is made up of 6 transmembrane segments; 1 to 4 form the voltage sensing domains (VSDs) and segments 5 and 6 of each domain form the central module of the pore. The β-subunits of sodium channels are composed of an N-terminal extracellular immunoglobulin-like fold, a single transmembrane segment, and a short intracellular segment as seen in **Figure 1** [5].

All sodium channel a-subunits consist of 4 homologous domains that form a single, voltage-gated aqueous pore. The a-subunits are greater than 75% identical over the aminoacid sequences comprising the transmembrane and extracellular domains. The -a-subunits show distinct patterns of expression, and are associated with accessory b-subunits which modify channel properties and interact with cytoskeletal and extracellular matrix proteins. Despite the broadly similar properties of voltage-gated sodium channels, there is good evidence for a specialised functional role of the various isoforms. Voltage-gated sodium channels provide the inward current that generates the upswing of an action potential in response to supra-threshold depolarisations of the membrane potential. At present 9 homologous channel-forming asubunits (Nav1.1 to Nav1.9), and four accessory b subunits (b1 to b4)) have been cloned from a variety of tissues and classified as belonging to the voltage-gated sodium channelfamily according to their amino acid sequence. Although theα-subunit alone is sufficient for the formation of a functional channel, the accessory β-subunits increase the efficiency of channel expression and are required for normal kinetics and voltage dependence of channel gating. In addition, β-subunits have an important role in the localization of α-subunits. The β4-subunit has even been shown to play a role in gating channels through a positively charged C-terminal region [6].

Mutations in the nine Nav channel subtypes (Nav1.1–Nav1.9) are associated with migraine, epilepsy, pain, and cardiac and muscle paralysis syndromes.

Specifically, the Nav1.7 channel is highly expressed in the soma, axons and peripheral endings of the nociceptive neurons of the spinal and trigeminal ganglia, in the olfactory neurons and in the neurons of the sympathetic ganglia. And to a lesser degree in the nervous system, liver, heart muscle and spinal cord.

Nav1.7 is designated as a threshold channel and is expressed on the surface of peripheral pain-sensitive neurons or nociceptors (in about 85%), where it conducts Na<sup>+</sup> currents in response to membrane depolarizations that are generated by potentially damaging events to the tissues, activating the action potential and sending pain signals. It can be said that it acts as a volume knob that sets the level of pain gain in the neurons. The Nav1.7 channel could also be seen as an amplifier of pain signals generated by receptors in nociceptor terminals.

Both gain and loss-of-function mutations in the SCN9A gene have been reported to result in a variety of conditions. When there is a gain of function, there is evidence of painful conditions such as hereditary erythromelalgia, paroxysmal extreme pain disorder, and idiopathic small fiber neuropathies. In contrast, loss-of-function mutations in the SCN9A gene have been reported to cause a rare disorder called congenital insensitivity to pain, characterized by the complete loss of the ability to feel pain stimuli.

The important role of Nav 1.7 in pain generation has created immense interest in this channel as an analgesic target.

#### **2.1 A little history**

The first hint that Nav1.7 might have an important role in pain signaling was given by a research group in 2004, who showed that three-generation patients from a Chinese family with a persistent pain syndrome called primary erythermalgia had missense mutations in the SCN9A gene, which codes for Nav1.7. Primary erythermalgia is a rare disease characterized by intermittent burning pain with redness and heat in the feet and hands in response to moderate exercise. In patients, standing, exercise, or local exposure to heat can induce the symptoms, and keeping the involved extremities at an icy cold temperature could be the only way to relieve the pain. The mutations in SCN9A that the group identified are located in the II/S5 segment (L858H) and the loop region between II/S4 and II/S5 (I848T) of Nav1.7 [7].

The next year, Dib-Hajj et al. discovered a third mutation in Nav1.7 in a large family with primary erythermalgia, they described a single substitution of phenylalanine by valine (F1449V) at codon 1449 in the sodium channel Nav1.7. This single amino acid substitution alters the biophysical properties of Nav1.7 and reduces the threshold for action potential firing and bursting of dorsal root ganglion neurons. This is what causes gain-of-function alterations in the channel, which causes hypersensitivity to pain [8].

Around the same time, researchers at University College London examined the role of Nav1.7 in pain pathways using knockout mice, which were found to be insensitive to inflammatory pain, showing that loss-of-function mutations abolish pain perception [9].

The first case of a patient with a congenital inability to perceive pain was said to been reported in the early twentieth century. Only very few patients have since been described and are categorized as having a congenital indifference to pain. In 2006 researchers at the University of Cambridge described individuals from three families with the extraordinary phenotype of a congenital inability to perceive any form of pain, in whom all other sensory modalities were preserved and the peripheral and central nervous systems were apparently otherwise intact. The index case for the study was a ten-year-old child, who acted in a "street theater" putting knives through his arms and later walking on hot coals without feeling any pain, reasons why he was well known to the medical service. He died at the age of 14, after jumping off a house roof, before being able to be part of the study. Subsequently, they studied three further consanguineous families in which there were individuals with similar histories of a lack of pain appreciation. The study included 6 children (between 6 and 12 years old), who had never felt any pain in any part of their body. Even as babies they had shown no evidence of pain appreciation. None knew what pain felt like,although the older individuals realized what actions should elicit pain(including acting as if in pain after football tackles). All had injuries to their lips (some requiring later plastic

surgery) and/or tongue (with loss of the distal third in two cases), caused by biting themselves in the first 4 years of life, fractures and even osteomyelitis; despite this, all children were found to have normal vision, hearing, and appearance. Detailed neurological examinations revealed that each could correctly perceive the sensations of touch, warm and cold temperature, proprioception, tickle and pressure, but not painful stimuli. Genome sequencing of these individuals showed disruption of one gene, SCN9A, causing loss-of-function mutations in Nav1.7 and complete loss of nociceptive input [1].

It is important to note that apart from the inability to feel pain, anosmia (loss of smell) is the only other sensory impairment in individuals with this channelopathy. Weiss et al examined human patients with loss-of-function mutations in SCN9A and show that they were unable to sense odours. To establish the essential role of Nav1.7 in odour perception, they generated conditional null mice in which Nav1.7 was removed from all olfactory sensory neurons. In the absence of Nav1.7, these neurons still produce odour-evoked action potentials but fail to initiate synaptic signalling from their axon terminals at the first synapse in the olfactory system. The mutant mice no longer display vital, odour-guided behaviours such as innate odour recognition and avoidance, short-term odour learning, and maternal pup retrieval. The no significant side effects in people lacking Nav1.7 such as cognitive, motor or non-nociceptive sensory impairments other than anosmia give support to the concept of Nav1.7 antagonists as analgesics [10].

These studies opened the way to propose and study the Nav1.7 channel as a therapeutic target to develop drugs that could serve as a new and potentially safer analgesic by selectively blocking it.

## **2.2 Why does the Nav1.7 channel represent a therapeutic target against pain?**

If the different subtypes of sodium channels are related to particular mechanisms of pain, then specific antagonists of those subtypes could, in theory, produce pain treatment without side effects.

Specifically, there are three binding sites that appear to offer the highest potential for the discovery and optimization of selective Nav1.7 inhibitors: 1) the extracellular vestibule of the pore, tetrodotoxin (TTX) and saxitoxin (STX) binding sites; 2) the extracellular loops of voltage-sensing domain II (VSD2) AND 3) the extracellular loops of voltage-sensing domain IV (VSD4).
