The external pore loop interacts with S6 and S3-S4 linker in domain 4 to assume an essential role in gating control and anticonvulsant action in the Na+ channel

Abstract

Carbamazepine, phenytoin, and lamotrigine are widely prescribed anticonvulsants in neurological clinics. These drugs bind to the same receptor site, probably with the diphenyl motif in their structure, to inhibit the Na+ channel. However, the location of the drug receptor remains controversial. In this study, we demonstrate close proximity and potential interaction between an external aromatic residue (W1716 in the external pore loop) and an internal aromatic residue (F1764 in the pore-lining part of the sixth transmembrane segment, S6) of domain 4 (D4), both being closely related to anticonvulsant and/or local anesthetic binding to the Na+ channel. Double-mutant cycle analysis reveals significant cooperativity between the two phenyl residues for anticonvulsant binding. Concomitant F1764C mutation evidently decreases the susceptibility of W1716C to external Cd2+ and membrane-impermeable methanethiosulfonate reagents. Also, the W1716E/F1764R and G1715E/F1764R double mutations significantly alter the selectivity for Na+ over K+ and markedly shift the activation curve, respectively. W1716 and F1764 therefore very likely form a link connecting the outer and inner compartments of the Na+ channel pore (in addition to the selectivity filter). Anticonvulsants and local anesthetics may well traverse this “S6 recess” without trespassing on the selectivity filter. Furthermore, we found that Y1618K, a point mutation in the S3-4 linker (the extracellular extension of D4S4), significantly alters the consequences of carbamazepine binding to the Na+ channel. The effect of Y1618K mutation, however, is abolished by concomitant point mutations in the vicinity of Y1618, but not by those in the internally located inactivation machinery, supporting a direct local rather than a long-range allosteric action. Moreover, Y1618 could interact with D4 pore residues W1716 and L1719 to have a profound effect on both channel gating and anticonvulsant action. We conclude that there are direct interactions among the external S3-4 linker, the external pore loop, and the internal S6 segment in D4, making the external pore loop a pivotal point critically coordinating ion permeation, gating, and anticonvulsant binding in the Na+ channel.

Exploring the Structure of the Voltage-gated Na+ Channel by an Engineered Drug Access Pathway to the Receptor Site for Local Anesthetics

“…These data confirm previous reports suggesting that the region at the interface between the P-loops of domains III and IV and the S6 segment of domain IV is a critical determinant of inactivation (23,(42)(43)(44)(45).…”

Gating transitions in the selectivity filter region of a sodium channel are coupled to the domain IV voltage sensor

“…Mutations in domain IV also specifically affect the binding of anticonvulsants to neuronal sodium channels (65). These drugs bind to the pore in an activity-dependent manner when applied to the external side but not to the internal side (66).…”

Loss of β1 accessory Na+ channel subunits causes failure of carbamazepine, but not of lacosamide, in blocking high‐frequency firing via differential effects on persistent Na+ currents

“…Identification of the location of the receptor site for CBZ on the Na + channel has been attempted in a number of studies. CBZ appears to share a receptor site with other anticonvulsant drugs such as lamotrigine and phenytoin (Ragsdale et al., 1996; Kuo et al., 2000; Kellinghaus, 2009), but experiments aimed at a precise identification of the binding site have been difficult to merge into a unifying hypothesis (see comprehensive discussion in Yang et al., 2009). A recent study has resolved some of the extant controversy by proposing that the common drug‐binding epitopes for CBZ, lamotrigine, and phenytoin involve structures in the external pore loop (W1716 in IVSS6), in the internal pore compartment of S6 in domain IV (F1764), as well as the S3‐4 linker of domain 4.…”