Inhibit, voltage-gated sodium channel leads to Altered kinetic of sodium channel

It is well known that ion channels are integral membrane proteins that are critical for the execution of action potential and therefore for neuronal function and activation. Action potentials are the electrical impulses that travel along the axons of neurons and result from the movement of Na⁺ and potassium (K⁺) ions across the membrane. Binding of excitatory neurotransmitters to their receptors opens cation-permeable ion channels causing the membrane to depolarise or become more positive. This depolarisation activates (opens) VGSCs allowing Na⁺ to enter the neuron further depolarising the membrane. This increase in membrane permeability to Na⁺ is responsible for the rising phase of the action potential, eventually causing the membrane polarity to reverse (overshoot phase). The falling phase of the action potential is caused by the inactivation of the VGSCs and the opening of voltage-gated potassium channels allowing K⁺ to leave the cell. The efflux of K⁺ ions results in hyperpolarisation (undershoot phase) of the membrane. Ultimately the voltage-gated K⁺ channels close and the membrane potential returns to its resting state. Type I and II pyrethroids cause stimulus dependent membrane depolarisation and conduction block.

It is therefore biologically plausible that binding of a chemical substance to a VGSC leads sodium channels to open at more hyperpolarised potentials and kept open longer (disruption of channel kinetic), allowing more sodium ions to cross and depolarise the neuronal membrane (Shafer et al., 2005)

Expression of VGSC are spatially and temporally dependent; however, it is biologically plausible that also in developing brain pyrethroids would bind to VGSC isoforms and disrupt the channel gating kinetic (Shafer et al., 2005; Soderlund et al., 2002).

Pyrethroids bind on the sodium channel α-subunit and affect nervous system function by altering their normal gating kinetics. Due to the extreme lipophilicity and the modest potency of pyrethroids radioligand, initial studies attempting to label the binding site were unsuccessful. The subsequent use of more potent radioligands were able to demonstrate high affinity saturable binding to brain sodium channels. However, the high lipophilicity of pyrethroids is still a limitation for the sensitivity of the assay and the identification of a single binding site on any given sodium channel and its mediated action (Soderlund et al., 2002; Trainer et al., 1997).

In hippocampal cell cultures from rat postnatal day 2–4 pups, patch clamp preparations of isolated neurons showed that deltamethrin alter the VGSC kinetic and inhibits neuronal activity in glutamatergic networks of hippocampal neurons in a potent and concentration-dependent manner (Meyer et al., 2008). Indeed, the actions of DLM are consistent with a decrease amplitude and number of spikes elicited using the current pulse (Meyer et al., 2008). In vitro exposure to pyrethroids (the type I permethrin and the type II deltamethrin) has been shown to differently disrupt sodium channel gate kinetics (Meyer et al., 2008) on hippocampal cultures from postnatal day 2–4 pups. This in vitro model was considered appropriate to explore effect on the developing brain. Cells were used for electrophysiological recording (patch clamp) 8–12 days in vitro (DIV) and hippocampal neurons isolated from early postnatal rodents form spontaneously active networks of interconnected neurons in which both glutamate and GABAergic neurotransmission occurs. Deltamethrin decreases neuronal excitability as measured by the rate of sEPSC activity at the concentration of 0.1 µM. At this concentration decrease in sEPSC interevent interval was rapid, occurring within 1–3 minutes of exposure and persistent, lasting throughout the exposure period (9 minutes). The effect on sEPSC frequency was concentration-dependent between 0.01 and 10 µM with an EC50 of 0.037 µM. There was no effect on the sEPSC amplitude at any tested concentration and this was consistent with previous data (Meyer and Shafer 2006), indicating that the effect does not include actions on postsynaptic glutamate receptors (Meyer et al., 2008).

DOSE CONCORDANCE

Although no evidence is available for the prototype stressor used in this AOP, deltamethrin, on the binding to VGSC, there is indirect evidence measuring the relationship between the MIE and the disruption of the VGSC gate kinetics. At concentration between 0.01 to 1 mM, deltamethrin has been shown to differently disrupt sodium channel gate kinetics (Meyer et al., 2008) on hippocampal cultures from postnatal day 2–4 pups. The effect was measured using the restricted patch clamp methodology and the results indicated that the observed change was concentration-dependent on the sEPSC without affecting the sEPSC amplitude and therefore excluding a postsynaptic excitatory mediated effect.

TIME CONCORDANCE

Changes in the VGSC kinetics are evident immediately following exposure in vitro to deltamethrin and recorded up to 9 minutes (Mayer et al., 2008)

The fact that binding of pyrethroids to VGSCs results in altered sodium channel gate kinetics is well accepted and supported by some evidence. However, some minor uncertainties can be detected as reported below.

Uncertainties in the overall knowledge remain as the sodium channels’ ontogeny is a complex process. Since brain development in both humans and rodents extends from early gestation through lactation it is not possible to state with certainty which isoform of the sodium channels’ α subunits is preferentially affected by deltamethrin.

For in vitro methodologies, there is still a lack of knowledge on stability of deltamethrin in the medium and the partitioning of this compound with plastic, lipid and protein. Indeed, the high lipophilicity of pyrethroids is still a limitation for the sensitivity of the assays and for the identification of a single binding site on any given sodium channel and its mediated action this may affect the sensitivity of the assays (Ruigt et al., 1987). Also, the metabolic competence of the test systems used in various assays is unknown.

Moreover, the study from Meyer et al. (2008) is an indirect measurement of the interaction between the prototype stressor, deltamethrin and VGSCs. Also, the exact temperature at which the patch clamp recording was made is uncertain (in the publication it is stated at room temperature) and it is well documented that pyrethroids effects on VGSCs are negatively temperature dependent (reviewed in Narahashi, 2000). Finally, Meyer and colleagues used hippocampal cell culture from rats PND 2–4 which were not characterised and did not contain microglia or oligodendrocyte precursors cells, therefore there are still uncertainties in the knowledge of the interaction between pyrethroids and microglia or oligodendrocytes precursor VGSC.

Some inconsistencies can be observed in experimental studies. They are associated with the electrophysiological technique used to study ionic currents in individual isolated living cells, tissue sections or patches of cells. The solution used in the bath can be similar to cytoplasm composition or completely different, they can be changed by adding ions or drugs to study the ion channels under different conditions. In the study of Meyer et al. (2008) different effects, i.e. burst duration, were observed for permethrin (type I) and deltamethrin (type II) and it was not clear if this represents a true difference in the mode of action between type I and type II pyrethroids or simply a difference between the two compounds. This could only be determined by the examination of additional chemicals.