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.