3.2 | Identification of the bupivacaine binding site in the central cavity and side pockets of Kv1.5 channels
The binding site of local anesthetics in the central cavity of Kv channels was never systematically mapped using an alanine mutagenesis screen. Strikingly, the data from the literature indicates that local anesthetics might also bind to residues outside the central cavity. Therefore, to characterize the bupivacaine binding site in detail, we performed a functional alanine scanning mutagenesis screen (59 mutants) of the pore forming S6 segment and the pore signature sequence, together with the S4 segment, the S4-S5 linker and the S5 segment, as these domains form the ‘side pockets´ of Kv1.5 channels (Marzian, Stansfeldet al. , 2013). To this end, the inhibition of bupivacaine for the different Kv1.5 alanine mutants was determined by voltage-clamp recordings in Xenopus oocytes. Note that endogenous alanine residues were mutated to valine. Under these experimental conditions, 250 µM bupivacaine caused an 81.0 ± 2.0 % (n = 8) inhibition of wild-type Kv1.5. The working hypothesis of this experimental approach was that a mutant channel lacking its regular amino acid side chain should have a reduced potency for bupivacaine. The potency of bupivacaine was significantly reduced for T480A of the pore signature sequence and for I502A, L510A, V512A and V516A of the S6 segment (Figure 1g). T480, V512 and V516 are pore facing amino acids belonging to the classical drug binding site for high affinity blockers in the central cavity of Kv1.5 channels (Decher, Kumar et al. , 2006; Decher, Pirard et al. , 2004; Marzian, Stansfeld et al. , 2013; Strutz-Seebohm, Gutcher et al. , 2007), whereas I502 faces into fenestrations connecting the central cavity with the side pockets and L510 directly faces into the ‘side pockets´ (Marzian, Stansfeld et al. , 2013). Moreover, we found that three residues (T479, T507 and V514) previously proposed to contribute to the local anesthetic binding site (Caballero, Moreno et al. , 2002; Franqueza, Longobardoet al. , 1997), did not significantly alter bupivacaine binding. In contrast, T480 of the pore signature sequence plays a much more pronounced role for local anesthetic binding than the initially proposed T479 (Caballero, Moreno et al. , 2002; Franqueza, Longobardoet al. , 1997). Thus, the alanine scan of the S6 segment already revealed revisited and also unexpected results for the binding site of the local anesthetic bupivacaine (Figure S1).
Most importantly, these results support the idea that bupivacaine interacts with the classical binding site in the central cavity and the selectivity filter as well as with parts of the ‘side pockets´ of the Kv1.5 channel. While we reported that mutations in the S4 and S4-S5 linker do not affect inhibition by bupivacaine (Figure 1g, dashed bars and box described by (Marzian, Stansfeld et al. , 2013)), the alanine scan of the S5 domain revealed significantly reduced bupivacaine potency for four mutant channels (Figure 1g). The four residues that we identified in S5, L436, F439, F440 and I443, directly face into the ‘side pockets´, similar as L510 of the S6 segment. These data further support that bupivacaine binds to the central cavity and the ‘side pockets´ of Kv1.5 channels.
As it was previously proposed that local anesthetics preferentially bind to the inactivated state of sodium channels, we tested for a correlation between inhibition and the intrinsic inactivation properties of the mutants. However, plotting the extent of C-type inactivation of single mutants against the respective inhibition, did not reveal such a correlation (Figure 1h). Thus, the residues identified by our alanine scan most likely exhibit a reduced affinity, due to an impaired drug binding.