Binding to VGSC leads to Altered kinetics of sodium channel

The biological plausibility for this KER is strong. It is a well-accepted fact that ion channels are integral membrane proteins that control the passage of various ions (Na+, K+, Ca2+, Cl−) across membranes in cells. The direction of ion transport through an open ion channel is governed by the electrochemical gradient for the particular ion species across the membrane in question. There is overwhelming evidence that binding of a chemical to a VGSC alters sodium channels kinetics. This is well supported by studies in which individual channel residues are mutated, and these mutations alter the ability of different chemicals to interact with the sodium channel to alter its gating kinetics (e.g. Vais et al., 2000; 2001). The stereospecific nature of effects of many different compounds on VGSC function further supports that specific binding leads to alterations in the kinetics of the channel (Soderlund 1985; Brown et al., 1988; Narahashi 1982).

The empirical evidence for this KER is strong. A wide variety of natural-occurring toxins have been demonstrated to interact with VGSCs and alter function of the channel. These toxins include TTX, the poison in fugu (pufferfish); scorpion and sea anemone toxins, brevotoxins from dinoflagellates, ciguatoxins and some conotoxins from fish-hunting snails. VGSCs possess six or more distinct receptor sites on the VGSC protein, and binding to each of these sites has differential effects on channel kinetics. For example, TTX binds at site 1, irreversibly blocking the pore of the channel and preventing sodium from moving through the channel. Brevotoxin binds to site 5, enhancing activation and preventing inactivation of the channel. The binding of delta conotoxin to site 6 slows channel inactivation (Catterall et al. 2007).

Ample knowledge is also available for synthetic pyrethroid insecticides which bind to the sodium channel α-subunit altering the normal gating kinetics of VGSC. Initial studies attempting to label the specific binding site of pyrethroids were unsuccessful due to the extreme lipophilicity and the modest potency of pyrethroid radioligands. The subsequent development of more potent radioligands demonstrated high affinity saturable binding to sodium channels in the brain. However, the high lipophilicity of pyrethroids still limited the sensitivity of the assay and obscured the identification of the single binding site responsible for pyrethroid action (Soderlund et al., 2002; Trainer et al., 1997). Despite these limitations, there remains overwhelming evidence that binding of pyrethroids to VGSC alters sodium channel kinetics. Mutations in the VGSC in insects alter gating kinetics by decreasing the sensitivity of the channel to pyrethroids, and provide resistance to their toxicity (Vais et al., 2000; 2001). In addition, the effects of pyrethroids are stereospecific where some isomers can interact with and modify channel function while other isomers are unable to bind and have no effect on channel kinetics (Soderlund 1985; Brown et al., 1988; Narahashi 1982). These properties of this class of insecticides on VGSCs are well established in the literature and have been extensively reviewed (Soderlund et al. 2002).

Dose and temporal concordance

Dose-dependent actions of pyrethroids on VGSC kinetics are well documented in the peer-reviewed literature (see Song and Narahashi, 1996, Tabarean and Narahashi, 1998). On the other hand, temporal concordance for this KER is difficult to measure because of the rapidity (msec) with which chemical binding to the VGSC changes the conformation of the channel and its gating kinetics. However, from the detailed biological understanding of the KER binding needs to precede the change in conformation and gating kinetics. In addition, there is clear evidence that the binding to VGSC by some pyrethroids is dependent on states of the channel (e.g. open, closed, activated, deactivated). In such cases, modification of the channel kinetics is “use-dependent”, i.e., activation is increased with subsequent stimuli that result in channel opening (Wu et al., 2021; Tabarean and Narahashi, 2001). This characteristic of use-dependence is strong evidence that binding to the channel affects its gate properties.

The fact that binding of chemicals to VGSCs results in altered sodium channel gate kinetics is well accepted and supported by abundant evidence. However, some minor uncertainties can be detected as reported below. Uncertainties in the overall knowledge remain; complete characterization of interactions of chemicals with all α isoforms of the channel, especially in mammals, as well as different subunit combinations have not been conducted, and differences likely exist based on different α and α/β subunit combinations. This is especially true for those channels that might be expressed during development, as the ontogeny of sodium channels is a complex process. Since brain development in both humans and rodents extends from early gestation well into the postnatal period it is not possible to state with certainty which isoform of the sodium channel’s α subunits is preferentially affected.

There are currently no quantitative models that predict the relationship between these KEs. However, it is possible to compute the population of VGSC that are affected by pyrethroid binding, and it has been estimated that less than 1% of the VGSC population (Narahashi et al., 1998) needs to be bound by pyrethroid to disrupt excitability in the neuron (KER2).

Chemicals may bind to VGSCs at various sites leading to different types of changes in the VGSC gate kinetics, and these changes also depend on the affinity of the chemicals to the binding sites (see section above, on KER description). Moreover, there are 9 different types of VGSCs including a complex ontology for the subunits. This complexity currently impedes the characterization of quantitative understanding.

The KER is active within milli-seconds and the upstream event occurs before the downstream event.

There are currently no known Feedforward/Feedback loops influencing this KER.