Sin selectividad, todos los grupos de tallas representados

Voltage-sensitive sodium channels are essential transmembrane proteins that are capable of generating and propagating of action potentials in order to conduct electrical signalling in neurons and other cells that are electrically excitable.

Voltage-sensitive sodium channels are characterized by their voltage-sensitive fast activating and immediately inactivating, and selective sodium ion conductance [88]. At resting conditions, sodium channels are closed. When the membrane depolarizes, sodium channels open, resulting in positively charged sodium ions entering the neuron and depolarizing the membrane potential. This initiates the uprising period of an action potential. After a few milliseconds, inactivation of the sodium channel occurs, and this prevents passage of sodium ions. This rapid inactivation generates, in part, the falling phase of the action potential [62, 88, 89].

During repolarization, the sodium channel deactivates, recovers from inactivation and completes the transition from the inactivated state to the closed resting state, ready for being opened by depolarisation [90].

Sodium channels in the mammalian central nervous system consist of a large α subunit that form the pore, associated with one or two smaller β subunits. There are nine different sodium channel genes (Nav1.1-Nav1.9) that have been have been

cloned, confirmed the functional expression, and characterized [88]. There is a tenth isoform (NaX) that has approximately 50% amino-acid sequence identical to the other mammalian sodium channels, but has not been functionally expressed [91].

The α subunits are organized in four homologous domains (DI-DIV), each of which is composed of six helical transmembrane segments named S1–S6. Segments S1-S4 form an important structural module, the voltage-sensing domain, which regulates the channel opening upon depolarization of membrane. The S4 segment in each domain contains a positively charged residue followed by two hydrophobic residues positioned within each S4 helix. Segments S5, S6, and P loops that connect S5-S6 from the four domains, form the pore domain [92]. The short intracellular loop that connects homologous domains III and IV functions as the inactivation gate, closing the channel pore from the inside during fast inactivation of the membrane [93].

Within the pore domain, each P-loop has five or six residues to form a selectivity filter (SF), a narrow vestibule that differentiate ions with similar charges and radii.

In mammalian sodium channels, the negatively charged of the asymmetric selectivity filter vestibule has two rings of highly conserved residues. There are four acidic residues (EEDD or EEMD) that constitute the outer ring of the SF and are related to the ion permeation rate. The inner ring or the constriction site of the SF, corresponding to conserved residues DEKA from DI-DIV, respectively, has been identified as the major determinant ion selectivity [93]

Two insect sodium channel genes, Vssc and DSC1, were isolated from the genome of Drosophila melanogaster [72, 94]. DSC1 was screened from the genome of Drosophila melanogaster using an eel sodium channel cDNA probe [94]. A DSC1 orthologous gene was later reported from the German cockroach (Blattella germanica) and Heliothis virescens [95]. DSC1 has been predicted to encode a sodium channel, given that amino acid sequence deduced of this gene and the overall domain organization is very similar to those of eel and mammalian sodium channels. However, the DSC1 protein sequence contain DKEA or DEEA instead

of the highly conserved DEKA motif, and preferentially conduct a novel type of voltage sensitive channel permeable to Ca ²⁺ [95]

Mutations in the voltage-sensitive sodium channels

The sodium channels are the primary molecular target of DDT and pyrethroid insecticides^. The binding of pyrethroids at their target sites in the VSSC inhibits the gating transition from activation (ion-conducting) to inactivation (non-conducting) state of action potentials [51]. Type I compounds prolong the opening duration of sodium channels, only long enough to include repetitive firing of action potentials.

Whereas type II pyrethroids hold the channel opening for such long period of time to modify membrane depolarization and cause conductance block of the nervous system. Voltage clamp studies in squid and crayfish giant axons have identified that pyrethroids modify sodium channels to remain open for an extended period of time.

This prolongs the sodium current during depolarization and slows the tail current associated with repolarization [54]. A transient sodium current induced by this prolongation, is associated with membrane depolarization and slowly decaying sodium tail current after repolarisation [54]. Patch clamp single-channel current experiments have demonstrated that pyrethroids delay the gating kinetics of both activation and inactivation gates [96]. More recent studies in Xenopus oocytes expressing sodium channels of insect and mammalian have confirmed that Type I pyrethroids modify resting or inactivated sodium channels; and in contrast, Type II pyrethroids preferentially bind to the activated (open) state of sodium channels [97].

The widespread, continued, and effective use of pyrethroids to control insect populations for prevention of insect-borne diseases has caused the development of resistance in many insect species. The most common pyrethroid resistance mechanism is point mutations in the voltage-sensitive sodium channel (Vssc) gene, that have been associated with insensitivity of the receptors to these products in the Vssc across the neuron [98]. This mechanism is called knockdown resistance (kdr), indicating the ability of insects to survive prolonged insecticides exposure without being ‘knocked-down’. The first evidence of knock down resistance trait was reported in DDT resistant strains of M. domestica [99]. This kdr trait confers

resistance to the rapid paralysis (knockdown) and lethal effect of DDT and pyrethroids. Subsequent studies identified an allelic form of kdr (termed super- kdr) that conferred a higher level of resistance to DDT and pyrethroids [98]. The kdr and super-kdr traits were mapped to a single gene on chromosome 3 in houseflies and reported to decrease sensitivity of the housefly central nervous system to DDT [100]. Further genetic linkage analysis demonstrated that kdr and super kdr mutations were linked to the para-orthologous sodium channel gene of the housefly (Vssc1 gene) [101]. Eventually, by comparing DNA sequences of sodium channel between several susceptible and kdr strains has confirmed that substitutions of L1014F within the domain IIS6 and M918T in the domain IIS4-S5 linker were responsible for kdr and super- kdr phenotypes respectively [102]. The first report of the L1014F mutation in M. domestica, and later in B. germanica has initiated the identification a series of divergent substitutions (C, H, S, or W) located at position 1014 in pyrethroid-resistant insect populations [103]. It seems that the L1014F, and its variants L1014H and L1014S generally contribute the moderate level of pyrethroid resistance (10–20-fold reduction in sensitivity) [99].

Figure 1.3 Drosophila V^SSC (voltage sensitive sodium channel) or para gen (A) Schematic presentation of the transmembrane arrangement of the main subunit (α)

of the sodium channel adopted as the general convention in most sodium channel gene descriptions. The S4 segments indicated by (+) are suggested to participate in the voltage sensing mechanism. The intramembrane short segments SS1 and SS2 are referred to as the pore region. The black triangles represent the entrance of the pore. The loop connecting domains III and IV is the region suggested to participate in the fast inactivation (B) Schematic presentation of the predicted membrane topology of the Drosophila para V^SSC α subunit. The approximate location of alternative exons is indicated. Taken from Zlotkin E: The insect voltage-gated sodium channel as target of insecticides. Annu Rev Entomol 1999, 44:429-55.

(Figure 1 p.432) [62].

Since first detected in the housefly, more than 50 pyrethroid resistant related mutations or combinations of mutations have been identified in sodium channels of populations of arthropod pests [90]. There were some mutations that can be observed in more than one species (Figure 1.4) whereas others have been detected only in a particular species (Figure 1.5) [103]. Expression in Xenopus laevis oocyte system has confirmed that 20 of these mutations cause insensitivity of insect sodium channels to pyrethroids, in Drosophila melanogaster (DmNaV); in B. germanica (BgNaV); in M. domestica (Vssc1); or in Ae. aegypti (Vssc) [103, 104]. The majority of mutations being confirmed to confer pyrethroid resistance are located clustered in or next to the intracellular linkers between transmembrane segments S4 and S5 (designated L4–5) of domain II; within transmembrane segments S5 and S6 of domain II; or within segment S6 of domain III [103]. The clusters of kdr mutations in these locations are in good agreement with computer modelling using a homology model of an open sodium channel of the housefly predicting the pyrethroid binding sites [105]. In this model, it was discovered that a hydrophobic pocket facing the lipid bilayer the S4–S5 linker and the S5 and S6 helices of domain II, together with the S6 helix of domain III, were found to shape [105]. The kdr mutations in these locations mostly confer resistance by reducing binding of pyrethroids to sodium channels. These structural models of pyrethroid binding sites in the lipid-exposed interface formed by segments IIIS6, IIS5 and linker segments IIS4–IIS5 [103] are also supported by additional data from systematic site directed mutagenesis studies that have identified more substitutions associated with reduced channel sensitivity.

Figure 1.4 Locations of sodium channel mutations that have been associated with pyrethroid resistance and detected in more than one species. Those kdr mutations which were confirmed in reducing the sodium channel sensitivity to pyrethroids in Xenopus oocytes are shown in filled circles. Empty circles indicate the mutations that have not been confirmed in Xenopus oocytes. The pore-forming α-subunit consists of a single polypeptide chain with four internally homologous domains (I–

IV), each having six transmembrane helices (S1–S6). Mutations are numbered according to the amino acid sequence of the housefly Vssc1 sodium channel protein (GenBank accession number: X96668). Taken from Rinkevich F, Rinkevich Y, Du K, Dong: Diversity and convergence of sodium channel mutations involved in resistance to pyrethroids. Pesticide biochemistry and physiology 2013, 106(3):93-100. (Fig. 1, p.94). [103].

Figure 1.5 Locations of sodium channel mutations that have been associated with pyrethroid resistance and detected only in one species. Those kdr mutations which were confirmed in reducing the sodium channel sensitivity to pyrethroids in Xenopus oocytes are shown in filled circles. Empty circles indicate the mutations that have not been confirmed in Xenopus oocytes. The pore-forming α-subunit consists of a single polypeptide chain with four internally homologous domains (I–

IV), each having six transmembrane helices (S1–S6). Mutations are numbered according to the amino acid sequence of the housefly Vssc1 sodium channel protein (GenBank accession number: X96668). Taken from Rinkevich F, Rinkevich Y, Du K, Dong: Diversity and convergence of sodium channel mutations involved in resistance to pyrethroids. Pesticide biochemistry and physiology 2013, 106(3):93-100. (Fig. 2, p.94). [103].

Mutation in voltage-sensitive sodium channels of Ae. aegypti

Various mutations in the sodium ion channel gene that give rise to resistance to pyrethroids in a number of insect species are also detected in dengue vector Ae.

aegypti. Although the most common L1014F mutation has not yet been identified in Ae. aegypti mosquitoes, a total of 12 new pyrethroid resistance-associated mutations has been detected [106]. These include the first identified kdr mutations:

L982W, V1016G, G923V and I1011M [107]; I1011V and V1016I in Latin America [108]; F1534C [109, 110], S989P [111] and D1763Y [112] in Asian populations, and T1520I [113] in India. Recently, the mutation V410L was identified in Brazil [114], and the mutation V419L in Colombia [115]. It has been functionally confirmed that five of these kdr mutations, S989P, I1011M, V1016G, F1534C, and recently V410L, reduce sodium channel sensitivity to pyrethroids [116].