Retigabine (RTG) is an anticonvulsant drug with a novel mechanism of action. It activates neuronal KCNQ-type K ϩ channels by inducing a large hyperpolarizing shift of steady-state activation. To identify the structural determinants of KCNQ channel activation by RTG, we constructed a set of chimeras using the neuronal K v 7.2 (KCNQ2) channel, which is activated by RTG, and the cardiac K v 7.1 (KCNQ1) channel, which is not affected by this drug. Substitution of either the S5 or the S6 segment in K v 7.2 by the respective parts of K v 7.1 led to a complete loss of activation by RTG. Trp236 in the cytoplasmic part of S5 and the conserved Gly301 in S6 (K v 7.2), considered as the gating hinge (Ala336 in K v 7.1), were found to be crucial for the RTG effect: mutation of these residues could either knockout the effect in K v 7.2 or restore it partially in K v 7.1/K v 7.2 chimeras. We propose that RTG binds to a hydrophobic pocket formed upon channel opening between the cytoplasmic parts of S5 and S6 involving Trp236 and the channel's gate, which could well explain the strong shift in voltage-dependent activation.

Cytoplasmic S4-S5 loops have been shown to be involved in fast inactivation of voltagegated ion channels. We studied mutations in these loops and their potential cooperative effects in domains D3 (N1151C, A1152C, I1160C/A) and D4 (F1473C, L1482C/A) of the human skeletal muscle Na + channel α-subunit (hNa v 1.4) using expression in tsA201 cells and the whole cell patch-clamp technique. All cysteine mutations were accessible to intracellularly applied sulfhydryl reagents which considerably destabilized fast inactivation. For different combinations of corresponding D3/D4 double mutations, fast inactivation could be almost completely removed. Thermodynamic cycle analysis indicated an additive effect for N1151C/F1473C and a significant cooperative effect for I1160/L1482 double mutations. Application of oxidizing reagents such as Cu-phenanthroline to link two cysteines via a disulfide bridge did not reveal evidence for a direct physical interaction of cysteines in D3 and D4. In addition to the pronounced alterations of fast inactivation, mutations of I1160 shifted steady-state activation in the hyperpolarizing direction and slowed the kinetics of both activation and deactivation. Sulfhydryl reagents had charge-dependent effects on I1160C suggesting interaction with negative charges in another protein region. We conclude that fast inactivation of the Na + channel involves both S4-S5 loops in D3 and D4 in a cooperative manner. D3/S4-S5 also plays an important role in activation and deactivation.