1 | INTRODUCTION
Local anesthetics block sodium channels by binding to the inactivated
state which provides the molecular basis to prevent pain perception.
However, local anesthetics also block diverse cardiac ion channels which
partially contributes to the cardiotoxicity of local anesthetics
(Castle, 1990; Clarkson & Hondeghem, 1985; Lipka, Jiang et al. ,
1998; Valenzuela, Delpon et al. , 1995). Bupivacaine for instance
is a long-acting local anesthetic which increases heart rate and
arterial blood pressure, reduces cardiac stroke volume and ejection
fraction, decreases conductivity and contractility and tends to induce
arrhythmias and a long QT syndrome (Clarkson & Hondeghem, 1985;
Kotelko, Shnider et al. , 1984; Sanchez-Chapula, 1988; Scott, Leeet al. , 1989).
Kv1.5 channels that generate the ultrarapid delayed rectifier currentI kur regulate atrial action potential durations
(Fedida, Wible et al. , 1993; Snyders, Tamkun et al. , 1993)
and are major drug targets for the treatment of atrial fibrillation
(Decher, Kumar et al. , 2006; Kiper, Rinné et al. , 2015).
Kv1.5 is blocked by bupivacaine in a potent and stereoselective manner
(Franqueza, Longobardo et al. , 1997; Valenzuela, Delpon et
al. , 1995). Voltage-gated ion channels share the common feature of a
water filled central cavity containing the classical drug binding site
in the inner mouth of the channel. The binding sites are mostly formed
by two to three amino acid residues of the pore forming helices and one
to two residues located in the pore helix. The position of the residues
involved in drug binding within these regions are highly conserved from
sodium over calcium to different potassium channels (Decher, Kumaret al. , 2006; Decher, Pirard et al. , 2004; Hanner, Greenet al. , 2001; Hockerman, Dilmac et al. , 2000; Mitcheson,
Chen et al. , 2000). The recently identified ‘side pockets´ in
voltage-gated potassium (Kv) channels formed by the backsides of the S5
and S6 segments together with the S4 and the S4-S5 linker of the
neighbouring subunit, serve as a drug binding pocket, providing the
molecular basis for an allosteric and irreversible Kv1 specific channel
inhibition (Marzian, Stansfeld et al. , 2013).
In early seminal studies probing the pore of Kv channels, it was
reported that mutations affecting T441 and T469 of the DrosophilaKv1-related Shaker channel alter open channel block of quaternary
ammonium compounds (Choi, Mossman et al. , 1993; Yellen, Jurmanet al. , 1991). Thus, open channel block was proposed to require,
as described above for many channels in detail, binding to two sites,
one located in the pore loop and one located in the inner mouth of the
channel (Baukrowitz & Yellen, 1996). The two residues identified in
these early studies correspond to Kv1.5 residues T479 in the pore
signature sequence and T507 of the S6 segment. Furthermore, L510 was
discussed as an important drug binding residue in Kv1.5, as studies with
the homologue Kv2.1 and Kv3.1 channels showed an altered pharmacology
for mutants corresponding to L510 (Aiyar, Nguyen et al. , 1994;
Shieh & Kirsch, 1994). These observations, together with a helical
wheel blot analyses led to the misinterpretation that T479, T507, L510
and V514 line the inner cavity of the Kv1.5 channel pore, forming the
drug binding site of the channel (Yeola, Rich et al. , 1996). The
subsequent studies investigated the role of these putatively pore facing
residues as possible binding sites for local anesthetics like
bupivacaine (Franqueza, Longobardo et al. , 1997) and benzocaine
(Caballero, Moreno et al. , 2002), but also rupatadine (Caballero,
Valenzuela et al. , 1999) and irbesartan (Moreno, Caballeroet al. , 2003). The crystal structure of the closely related
rKv1.2 channels (Long, Campbell et al. , 2005) revealed however
that the amino acids T507, L510 and V514 are not pore facing. In
contrast, T507 and L510 perfectly face into the recently identified
‘side pockets´ that play a crucial role for the development of Kv1
specific blockers (Marzian, Stansfeld et al. , 2013). This led to
the question whether bupivacaine and other local anesthetics exclusively
interact with the central cavity or also require interactions with the
newly identified ‘side pockets´ (Marzian, Stansfeld et al. ,
2013).
To address this question we mapped the binding site of the two local
anesthetics bupivacaine and ropivacaine using a systematic functional
alanine scanning mutagenesis screen of the S4, S4-S5, S5 and the S6
segments of Kv1.5, combined with in silico docking experiments
and molecular dynamics simulations. 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. In addition, we found that a binding of local anesthetics
to the central cavity and the ‘side pockets´ is conserved while the
binding modes show considerable variations which might provide the
molecular basis to modulate specificity, stereoselectivity and thus the
side effects of local anesthetics.