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.