A Single Residue in the S6 Transmembrane Domain Governs the Differential Flecainide Sensitivity of Voltage-Gated Potassium Channels

Abstract

Flecainide has been used to differentiate Kv4.2-based transient-outward K ϩ -currents (flecainide-sensitive) from Kv1.4-based (flecainide-insensitive). We found that flecainide also inhibits ultrarapid delayed rectifier (I Kur ) currents in Xenopus laevis oocytes carried by Kv3.1 subunits (IC 50 , 28.3 Ϯ 1.3 M) more strongly than Kv1.5 currents corresponding to human I Kur (IC 50 , 237.1 Ϯ 6.2 M). The present study examined molecular motifs underlying differential flecainide sensitivity. An initial chimeric approach pointed to a role for S6 and/or carboxyl-terminal sites in Kv3.1/Kv1.5 sensitivity differences. We then looked for homologous amino acid residues of the two sensitive subunits (Kv4.2 and Kv3.1) different from homologous residues for insensitive subunits (Kv1.4 and Kv1.5). Three candidate sites were identified: two in the S5-S6 linker and one in the S6 segment. Mutation of the proximal S5-S6 linker site failed to alter flecainide sensitivity. Mutation at the more distal site in Kv1.5 (V481L) modestly increased sensitivity, but the reciprocal Kv3.1 mutation (L401V) had no effect. S6 mutants caused marked changes: flecainide sensitivity decreased ϳ8-fold for Kv3.1 L422I (IC 50 , 213 Ϯ 9 M) and increased ϳ7-fold for Kv1.5 I502L (IC 50 , 35.6 Ϯ 1.9 M). Corresponding mutations reversed flecainide sensitivity of Kv1.4 and Kv4.2; L392I decreased Kv4.2 sensitivity by ϳ17-fold (IC 50 of 37.4 Ϯ 6.9 to 628 Ϯ 36 M); I547L increased Kv1.4 sensitivity by ϳ15-fold (IC 50 of 706 Ϯ 37 to 40.9 Ϯ 7.3 M). Our observations indicate that the flecainide sensitivity differences among these four voltagegated K ϩ -channels are determined by whether an isoleucine or a leucine is present at a specific amino acid location.

The Molecular Basis of High-Affinity Binding of the Antiarrhythmic Compound Vernakalant (RSD1235) to Kv1.5 Channels

“…Similar experiments were carried out for flecainide (Fig. 4A), and in qualitative agreement with previous oocyte data from Val481, the nearby residue in the selectivity filter (Herrera et al, 2005), the mutation of Thr479 to alanine increased the potency of flecainide (Fig. 4 -8).…”

“…3) were obtained for the reduced block mutants, I502A, V505A, and I508A, in comparison with a positive control mutation that increased block (T479A), and for a readily available comparative antiarrhythmic agent, flecainide (Fig. 4), whose block is also reported to be affected by mutations in the lower S6 (Herrera et al, 2005). Vernakalant data in Fig.…”

“…1 suggest that the S6 domain that lines the inner vestibule of the channel and the deep pore near the selectivity filter are likely areas where vernakalant could bind to Kv1.5. Extensive studies of some approved and developmental antiarrhythmic agents that target either the cardiac Na ϩ channel or K ϩ channels involved in action potential repolarization have suggested that the base of the selectivity filter in the deep pore and the S6 are important areas for antiarrhythmic drug binding within the ion conduction pathway (Decher et al, 2004;Herrera et al, 2005). These regions were targeted for mutational analysis of block by replacement of WT residues with alanines wherever possible, valines if the WT residue was an alanine, and leucine, valine, or glycine if there was an expression problem with alanine.…”

“…Flecainide is also a drug of choice for the acute conversion of atrial fibrillation to sinus rhythm (Fuster et al, 2006) and is known to block Kv1.5 (Grissmer et al, 1994;Herrera et al, 2005) but with much lower potency than vernakalant. In the present study, we have investigated the binding site of vernakalant in the deep pore and S6 of Kv1.5 using both electrophysiology and site-directed mutagenesis.…”

“…The potency of these agents is affected by introduction of specific mutations in the S6 domain of Kv1.5, which lines the inner vestibule of the channel (Val505,Thr507,Ile508,Leu510,Val512,Val514), or mutations in the deep pore (Thr479 and Thr480) near the selectivity filter (Yeola et al, 1996;Franqueza et al, 1997;Caballero et al, 2002;Decher et al, 2004Decher et al, , 2006Herrera et al, 2005). Many of these same residues are also important sites of interaction for the Kv␤ inactivation particle (Decher et al, 2005), for tetraethylammonium (Choi et al, 1993;Lopez et al, 1994), and for 4-aminopyridine block (Kirsch and Drewe, 1993).…”