Local anesthetic block of Kv channels:: Role of the S6 helix and the S5-S6 linker for bupivacaine action

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 (K-d between 110 and 240 muM), and a time-independent block on Kv2.1 (K-d = 220 muM). At 32 degreesC, 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 (K-d increased from 220 to 1170 muM). 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.