SCBIs Act at Site 10 on the Na Channel

The voltage-dependent Na+ channel blocking action of pyrazolines was immediately recognized as being similar to that of local anesthetics, class I anticonvulsants, and class I antiarrhythmics (Salgado, 1992), a structurally broad range of drugs (Clare et al., 2000; Anger, 2001) all known to act at a common blocker site within the Na+ channel pore. Consistent with the nomenclature of Soderlund (see Chapter 5.1), this will be called site 10. Like the SCBIs, drugs acting at site 10 all exhibit voltage dependence of block, deriving from selective binding to open and inactivated channel states.

Drugs acting at site 10 displace the binding of the radioligand [3H]batrachotoxin (BTX)-B from the BTX binding site in the Na+ channel (Postma and Catterall, 1984; Catterall, 1987). The interaction is clearly allosteric, because local anesthetics can block BTX-modified open channels without displacing the toxin (Wasserstrom et al., 1993; Zamponi et al., 1993), although they speed the dissociation rate of BTX from its binding site. Pyrazo-lines were also shown to potently displace specific binding of [3H]BTX-B (Deecher et al., 1991; Salgado, 1992). Payne et al. (1998) further examined the interaction between the pyrazoline RH-3421 and the local anesthetic dibucaine in BTX binding studies. Each of these compounds decreased the potency of the other as an inhibitor of BTX binding approximately as much as expected from the assumption that they share a common binding site. Furthermore, RH-3421 increased the dissociation rate of [3H]BTX-B from its binding site, also expected from an action at site 10 (Deecher etal, 1991).

Lapied et al. (2001) examined the interactions of DCJW with the local anesthetic lidocaine and the guanidinium blocker tetrodotoxin, which is known to block the pore from the external face, at a binding site distinct from the local anesthetics (see Chapter 5.1). The IC50 for DCJW was not affected by the presence of an IC50 concentration of tetrodotoxin (TTX) in the external solution, consistent with independent action of the two compounds at distinct binding sites. In the presence of an IC50 concentration of lidocaine, however, the IC50 for DCJW was increased about 30-fold. A twofold shift in equilibrium binding would be expected from the hypothesis that both compounds act at the same site (Cheng and Prusoff, 1973), so the mechanism of the observed 30-fold shift is not fully understood. Nevertheless, available evidence is consistent with the action of the SCBIs at site 10.

Studies on permanently charged quaternary derivatives of local anesthetics were crucial in localizing site 10 within the Na+ channel pore. Being permanently charged, these molecules cannot cross membranes and are therefore not clinically useful. However, when applied within the cell, they are able to enter the channel from the internal mouth, only when the channel is open, giving rise to a phenomenon known as use dependence - increase in the number of blocked channels with stimulation. Because the permanently charged quaternary molecule cannot fit through the ion-selective constriction in the channel known as the selectivity filter, it remains trapped within the channel after activation gate closure. Alternatively, more lipophilic compounds acting at site 10, when caught within the closed channel, can diffuse out laterally through lipophilic pathways in the channel wall. SCBIs behave like local anesthetics with very slow kinetics, and do not display use dependence (Zhao et al., 2003), suggesting that the insecticides can only access the binding site from the lipophilic pathway and not through the internal mouth of the pore.

In addition to modulation of blocker access to the binding site by the activation gate, channel gating also modulates the equilibrium binding affinity of the channel for the molecules. Like the pyrazolines and DCMP/DCJW, there is little affinity of the blocker molecule for the resting state, but strong binding to the open and inactivated states. The bound molecule is thought to lie in a hydrophobic pocket that can be reached via either the aqueous or hydrophobic pathways. When bound, the molecule is thought to lie within the pore and block ion flow through it. Several excellent reviews on the interaction of blocking drugs with Na+ channels have recently been published (Clare et al., 2000; Anger et al., 2001; Wang and Wang, 2003).

The fact that blockers acting at site 10 bind strongly to a number of states after channel opening but not to the resting state, indicates that the movements of the S6 segments (see Section associated with channel activation lead to formation of the receptor for these compounds. The subsequent fast and slow inactivation steps are thought to involve processes at the internal membrane face that involve blocking of the pore by a so-called inactivation particle, presumably without further significant changes in the conformation of site 10 (see Chapter 5.1).

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