TY - JOUR T1 - Local Anesthetic Block of Kv Channels: Role of the S6 Helix and the S5-S6 Linker for Bupivacaine Action JF - Molecular Pharmacology JO - Mol Pharmacol SP - 1417 LP - 1429 DO - 10.1124/mol.63.6.1417 VL - 63 IS - 6 AU - Johanna Nilsson AU - Michael Madeja AU - Peter Århem Y1 - 2003/06/01 UR - http://molpharm.aspetjournals.org/content/63/6/1417.abstract N2 - To gain insights in the molecular mechanisms of anesthesia, we analyzed the effects of bupivacaine on a series of voltage-gated K+ channels (Kv1.1, -1.2, -1.5, -2.1, -3.1, and -3.2) and various mutant channels derived from Kv2.1, using Xenopus laevis oocytes. Two phenomenologically different blocking effects were seen at room temperature: a time-dependent block of Kv1 and Kv3 channels (Kd between 110 and 240 μM), and a time-independent block on Kv2.1 (Kd = 220 μM). At 32°C, however, Kv2.1 also showed a time-dependent block. Swapping the S6 helix between Kv1.2 and Kv2.1 introduced Kv1.2 features in Kv2.1. Critical residues were located in the N-terminal end of S6, positions 395 and 398. The triple substitution of residues 372, 373, and 374 in the S5-S6 linker decreased the bupivacaine affinity by 5-fold (Kd increased from 220 to 1170 μM). The results suggest that bupivacaine blocks Kv channels by an open-state–dependent mechanism and that Kv2.1 deviates from the other channels in allowing a partial closure of the channel with bupivacaine bound. The results also suggest that the binding site is located in the internal vestibule and that residues in the descending P-loop and the upper part of S6 are critical for the binding, most likely by allosteric mechanisms. A simple mechanistic scenario that explains the observations is presented. Thermodynamic considerations suggest that the interaction between bupivacaine and the channels is hydrophobic. ER -