Complexes of Peptide Blockers with Kv1.6 Pore Domain: Molecular Modeling and Studies with KcsA-Kv1.6 Channel

Abstract

Potassium voltage-gated Kv1.6 channel, which is distributed primarily in neurons of central and peripheral nervous systems, is of significant physiological importance. To date, several high-affinity Kv1.6-channel blockers are known, but the lack of selective ones among them hampers the studies of tissue localization and functioning of Kv1.6 channels. Here we present an approach to advanced understanding of interactions of peptide toxin blockers with a Kv1.6 pore. It combines molecular modeling studies and an application of a new bioengineering system based on a KcsA-Kv1.6 hybrid channel for the quantitative fluorescent analysis of blocker-channel interactions. Using this system we demonstrate that peptide toxins agitoxin 2, kaliotoxin1 and OSK1 have similar high affinity to the extracellular vestibule of the K-conducting pore of Kv1.6, hetlaxin is a low-affinity ligand, whereas margatoxin and scyllatoxin do not bind to Kv1.6 pore. Binding of toxins to Kv1.6 pore has considerable inverse dependence on the ionic strength. Model structures of KcsA-Kv1.6 and Kv1.6 complexes with agitoxin 2, kaliotoxin 1 and OSK1 were obtained using homology modeling and molecular dynamics simulation. Interaction interfaces, which are formed by 15-19 toxin residues and 10 channel residues, are described and compared. Specific sites of Kv1.6 pore recognition are identified for targeting of peptide blockers. Analysis of interactions between agitoxin 2 derivatives with point mutations (S7K, S11G, L19S, R31G) and KcsA-Kv1.6 confirms reliability of the calculated complex structure.

N-Terminal Tagging with GFP Enhances Selectivity of Agitoxin 2 to Kv1.3-Channel Binding Site

“…Interactions of GFP-L2-AgTx2 with the pore blocker binding sites of the Kv1.x (x = 1, 3, 6) channels were studied with the bioengineering systems based on E. coli spheroplasts that expose hybrid potassium channels KcsA-Kv1.x (x = 1, 3, 6) in their inner membrane. KcsA-Kv1.x (x = 1, 3, 6) hybrid channels bear extracellular binding sites of the corresponding eukaryotic Kv1 channels and provide both recognition of Kv1 channels pore blockers and evaluation of their affinity [15][16][17][18][19]. As systematically verified, data obtained with KcsA-Kv1.x (x = 1, 3, 6) for small organic compounds, peptide toxins, as well as fluorescent protein-tagged pore blockers are in agreement with the results of electrophysiological studies [12,16].…”

Molecular Dynamics Simulation Reveals Specific Interaction Sites between Scorpion Toxins and Kv1.2 Channel: Implications for Design of Highly Selective Drugs

“…About building the complexes of toxins and channels, some groups choose ZDOCK or HADDOCK molecular docking software to obtain the complexes [ 20 , 40 , 41 ]. Since the structure of ChTx and K v 1.2–2.1 chimera resolved, several groups use this resolved structure to build scorpion toxins and K + channel complexes [ 42 , 43 ]. Nekrasova et al performed homology modeling of K v 1.6-toxin complexes instead of molecular docking to obtain a more reliable model [ 43 ].…”

“…Since the structure of ChTx and K v 1.2–2.1 chimera resolved, several groups use this resolved structure to build scorpion toxins and K + channel complexes [ 42 , 43 ]. Nekrasova et al performed homology modeling of K v 1.6-toxin complexes instead of molecular docking to obtain a more reliable model [ 43 ]. Therefore, we choose ChTx and K v 1.2–2.1 chimera to build our toxin-channel complexes.…”

“…Kohl et al chose the structure with the highest number of H-bonds between the toxin and the channel [ 42 ]. Nekrasova et al chose 70 trajectory frames to analyse the hydrophobic interaction, the hydrogen, and the ionic bonds of the toxin [ 43 ]. Also, Yi et al analysed the last structure of the toxin-channel structure [ 20 , 40 ].…”

Fluorescent Ligands of Kv1 Channels on the Basis of Hongotoxin: Atto488-Hongotoxin

“…These KcsA-Kv1.x (x=1, 3, 6) expressed in E.coli cells embed into cell membrane, expose ligand-binding sites into periplasmic space and bind ligands when cells are transformed to spheroplasts. Measurement and quantitative treatment of fluorescence signals associated with spheroplasts in the presence of A-HgTx1 was performed using confocal laser scanning microscopy as described elsewhere [4][5][6].…”