4 | DISCUSSION
We found that binding to the central cavity and ‘side pockets´ is
conserved among local anesthetics, while in the ‘side pockets´ binding
can occur in different modes. Nevertheless, the local anesthetics bound
to the newly identified sites in the ‘side pockets´ are essentially
determining the potency of the drugs, as mutations in the ‘side pockets´
strongly diminish the affinity of both compounds, bupivacaine and
ropivacaine. Besides tuning the apparent affinity, the ‘side pockets´
are contributing to the enantioselectivity present for the inhibition of
Kv1.5 by bupivacaine. The regular Hill factor of the dose-response curve
of around one for the Kv1.5 inhibition indicates that local anesthetics
must independently bind to the central cavity and the ‘side pockets´,
with no cooperativity between the two sites. Yet, for an efficient drug
block or in the case of bupivacaine for a stereoselective inhibition,
binding to both sites, the central cavity and the ‘side pockets´,
appears mandatory. How many of the four ‘side pockets´ need to be
occupied by local anesthetic for an efficient channel inhibition remains
however an open question. This question is hard to address
experimentally, especially as we found that designing concatameric
channels often results in altered biophysical and pharmacological
properties.
Kv1.5 open channel blockers expose varying voltage- and use-dependencies
or cooperativity of inhibition. Tikhonov & Zhorov hypothesized that
this might be caused by a common mechanism, meaning that the compounds
either form a blocking particle itself by a charged moiety of the drug
or by binding of neutral drugs to a potassium at the S5site in the cavity, using different stoichiometries for the formation of
the respective blocking particle complex (Tikhonov & Zhorov, 2014).
From this common position underneath the selectivity filter
(S5 site) hydrophobic parts of the drugs were proposed
to either remain in the central cavity or to laterally protrude into the
side fenestrations to interact with I502, a residue relevant for many
Kv1.5 blockers. However, we now found that not only Psora-4, but also
ropivacaine and bupivacaine bind to the ‘side pockets´ to reach I502
from the other side of the fenestrations. Considering the current study,
several drugs were now reported to utilize the ‘side pockets´ to alter
drug affinity or in the case of bupivacaine also stereoselectivity.
Therefore, the discussed variabilities in the kinetics or cooperativity
of Kv1 channel inhibition might not be exclusively caused by the
formation of different charged drug potassium complexes in the central
cavity, but also or even exclusively by an additional drug binding in
the ‘side pockets´.
An open question in the field is how local anesthetics cause a
stereoselective inhibition of Kv1.5 channels. Kvß1.3 which binds to the
central cavity of Kv1.5 (Decher, Gonzalez et al. , 2008) reduces
stereoselectivity of bupivacaine inhibition (Arias, Guizy et al. ,
2007). Strikingly, the θ value is only reduced from about nine to four
(Arias, Guizy et al. , 2007), despite that Kvß1.3 interacts with
all the directly pore facing residues that we have also identified as
binding sites for local anesthetics, including T480, I508, V512 and V516
(Decher, Kumar et al. , 2005). These data indicate that there are
other residues outside the central cavity that co-determine the
stereoselectivity of Kv1.5 inhibition by local anesthetics.
Consistently, our mutagenesis data with T480A, F440A and I443A indicate
that stereoselective inhibition of Kv1.5 by bupivacaine is determined by
residues in the pore and the ‘side pockets´, also further supporting
that efficient inhibition by local anesthetics actually requires binding
to both distinct binding sites. For local anesthetics it has been shown
that reducing the length of the alkyl side chain at the piperidine ring
reduces affinity and stereoselectivity (Longobardo, Delpon et
al. , 1998). Strikingly, ropivacaine, which has a propyl instead of a
butyl side chain compared to bupivacaine and is exhibiting a reduced
affinity and almost no stereoselectivity for Kv1.5 inhibition
(Longobardo, Delpon et al. , 1998), actually maps to a different
binding site in the ‘side pockets´ than bupivacaine. The ropivacaine
binding site determined by in silico docking experiments and MD
simulations involves interactions with the S4 segment and the proximal
S4-S5 linker and a binding to the S5 segment of a neighboring channel
subunit. At the ropivacaine binding site, the interactions with the S5
segment residues F440 and I443 that are involved in determining the
stereoselective inhibition of bupivacaine, can easily be accomplished
and thus these residues are presumably accessible for both enantiomers.
In contrast, bupivacaine maps to the other site of the ‘side pockets´
from where it appears that an interaction with the S5 residues F440 and
I443 from the same subunit might be more restricted and only possible or
preferred for one of the enantiomers. The differential set of residues
that we have mapped for the two local anesthetics in the ‘side pockets´
provides the basis for future studies to carefully elaborate how binding
to the ‘side pockets´ contributes to stereoselective channel inhibition
of local anesthetics. Unfortunately, the above mentioned hypothesis
currently remains unaddressed as in silico docking experiments
and MD simulations are methodologically not powerful enough to resolve
how this relative small stereoselectivity is achieved for a blocker that
also displays a rather low potency of channel inhibition.
In earlier studies, it was thought that T479 of the pore signature
sequence, together with T507, L510 and V514 of the S6 segment face the
inner pore of Kv1.5 (Yeola, Rich et al. , 1996), as mutations at
these sites altered the pharmacology of the channel. The crystal
structures of the bacterial rKv1.2 channel (Long, Campbell et
al. , 2005) revealed that T507, L510 and V514 are not pore facing and
instead face into ‘side pockets´ that we have recently described as drug
binding site for the Kv1 channel blocker Psora-4 (Marzian, Stansfeldet al. , 2013). Consistently, we have identified in an
alanine-scanning approach a novel binding site for local anesthetics
which is located outside of the central cavity. Unfortunately, most
previous studies used only these limited set of mutants to analyze the
putative drug binding sites of quinidine (Yeola, Rich et al. ,
1996), benzocaine (Caballero, Moreno et al. , 2002), bupivacaine
(Caballero, Moreno et al. , 2002; Franqueza, Longobardo et
al. , 1997), rupatadine (Caballero, Valenzuela et al. , 1999) or
irbesartan (Moreno, Caballero et al. , 2003). Therefore, it is
possible that besides Psora-4 and local anesthetics, many more drugs,
including the ones mentioned above, are actually utilizing the ‘side
pocket´ to cause or modulate Kv1 channel inhibition.
Our results reveal that local anesthetics do not exclusively bind to the
central cavity and that binding to the ‘side pockets´ is essential for
the action of local anesthetics, providing the molecular basis to
modulate specificity, stereoselectivity and thus the spectrum of side
effects of local anesthetics.