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.