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Local Anesthetic Block of Kv Channels: Role of the S6 Helix and the S5-S6 Linker for Bupivacaine Action

The Nobel Institute for Neurophysiology, Department of Neuroscience, Karolinska Institutet, Stockholm, Sweden (J.N., P.Å.); and Institut für Physiologie, Münster, Germany (M.M.).

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 µM), and a time-independent block on Kv2.1 (K_d = 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 (K_d 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.

Address correspondence to: Peter Århem, The Nobel Institute for Neurophysiology, Department of Neuroscience, Karolinska Institutet, SE-171 77 Stockholm, Sweden. E-mail: peter.arhem{at}neuro.ki.se

This article has been cited by other articles:

				J. Nilsson, M. Madeja, F. Elinder, and P. Arhem Bupivacaine Blocks N-Type Inactivating Kv Channels in the Open State: No Allosteric Effect on Inactivation Kinetics Biophys. J., December 1, 2008; 95(11): 5138 - 5152. [Abstract] [Full Text] [PDF]