Figure legends
FIGURE 1 . Characterization of the inhibition of Kv1.5 channels by bupivacaine and mapping of the binding site. (a) Representative voltage-clamp recordings of Kv1.5 expressed in Xenopus laevisoocytes before and after application of 250 µM R/S-bupivacaine. (b) Dose-response curve for Kv1.5 inhibition by R/S-bupivacaine. (c) G/V-relationship of Kv1.5 in the absence and presence of 250 µM R/S-bupivacaine, respectively. (d) Analyses of the voltage-dependence of Kv1.5 inhibition by 250 µM R/S-bupivacaine in the voltage range of -20 to +70 mV. (e) Analyses of the use-dependence of Kv1.5 inhibition by R/S-bupivacaine (250 µM). The inset illustrates representative Kv1.5 currents measured before and the first two pulses directly after a 12 min pulse free period, during which the drug was washed-in while the cells were held at ‑80 mV. (f) Ratio of I drug toI control determined for the first voltage-step, derived from (e). (g) Alanine-scanning of the S4 segment, the S4-S5 linker, as well as the S5 and S6 segments. Analyses of the inhibition by 250 µM R/S-bupivacaine for the mutants and the respective wild-type channels. The values illustrated for the inhibition of S4 segment and S4-S5 linker mutants (dashed bars and box) was previously reported by Marzian et al. , 2013. (h) Lack of correlation between the inactivation properties of the mutants tested and their apparent affinity. The inactivation properties of the mutants were analyzed as percentage of inactivation over a time course of 1 s at +40 mV and were derived from the study by Marzian et al. , 2013. Significances are indicated with *, P < 0.01.
FIGURE 2. Stereoselectivity of Kv1.5 inhibition by bupivacaine is determined by residues in the central cavity and the ‘side pockets´. (a) Whole-cell patch clamp recordings of wild-type Kv1.5 recorded in HEK-293 cells, before and after the application of 50 µM R-bupivacaine or S-bupivacaine, respectively. (b) Patch clamp recordings of I443A, before and after the application of 250 µM R-bupivacaine or S-bupivacaine, respectively. (c-e) Dose-response curves of Kv1.5 inhibition by R-bupivacaine and S-bupivacaine for the (c) F440A, (d) I443A and (e) T480A mutants. Dashed lines indicate the respective dose-response curves for wild-type Kv1.5 inhibition by R-bupivacaine and S-bupivacaine described in the literature by Arias et al. , 2007. (f) Degree of the stereoselectivity (θ value) for wild-type Kv1.5 and the F440A, I443A and T480A mutants.
FIGURE 3 . Characterization of the inhibition of Kv1.5 channels by ropivacaine and mapping of the binding site. (a) Representative voltage-clamp recordings of Kv1.5 expressed in Xenopus laevisoocytes before and after application of 1 mM ropivacaine. (b) Dose-response curve for Kv1.5 inhibition by ropivacaine. (c) G/V-relationship of Kv1.5 in the absence and presence of 1 mM ropivacaine, respectively. (d) Analyses of the voltage-dependence of Kv1.5 inhibition by 1 mM ropivacaine in the voltage range of -20 to +70 mV. (e) Analyses of the use-dependence of Kv1.5 inhibition by 1 mM ropivacaine. The inset illustrates representative Kv1.5 currents measured before and the first two pulses directly after a 12 min pulse free period, during which the drug was washed-in while the cells were held at ‑80 mV. (f) Ratio of I drug toI control determined for the first voltage-step, derived from (e). (g) Alanine-scanning of the S4 segment, the S4-S5 linker, as well as the S5 and S6 segments. Analyses of the inhibition by 1 mM ropivacaine for the mutants and the respective wild-type channels. (h) Lack of correlation between the inactivation properties of the mutants tested and their apparent affinity. The inactivation properties of the mutants were analyzed as percentage of inactivation over a time course of 1 s at +40 mV and were derived from the study by Marzianet al. , 2013. Significances are indicated with *, P < 0.01.
FIGURE 4. Binding modes of S-bupivacaine and ropivacaine in a Kv1.5 homology model. (a) Top and side view of a Kv1.5 homology model based on the Kv1.2-Kv2.1 chimera crystal structure (PDB code: 2R9R). (b,c) Illustration of the binding mode of (b) S-bupivacaine and (c) ropivacaine in the central cavity of Kv1.5. Depicted are the most populated clusters determined by 100 ns molecular dynamics simulations. (d,e) Illustration of the most populated cluster or binding mode in the ‘side pockets´ for (d) S-bupivacaine and for (e) ropivacaine. (f,g) Illustration of the second most populated cluster or binding mode in the ‘side pockets´ for (f) S-bupivacaine and for (g) ropivacaine.
FIGURE 5. Differential binding mode of bupivacaine and ropivacaine in the ‘side pockets´ of Kv1.5. (a) Top view of a Kv1.5 homology model based on the Kv1.2-Kv2.1 chimera crystal structure (PDB code: 2R9R) with S-bupivacaine and ropivacaine bound to the four different ‘side pockets´. (b) Side view illustrating the differential binding modes of bupivacaine and ropivacaine. Neighbouring subunits can be distinguished as they are illustrated in gray versus dark gray.