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

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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.

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Abbreviations

AgTx2:

agitoxin-2

CLSM:

confocal laser scanning microscopy

IPTG:

isopropyl β-D-1-thiogalactopyranoside

KTx:

kaliotoxin 1

Kv:

voltage-gated potassium channel

MgTx:

margatoxin

MD:

molecular dynamics

OSK1:

a toxin from Orthochirus scrobiculosus scorpion venom

R-AgTx2:

agitoxin-2 labeled with 5(6)-carboxytetramethylrhodamine N-succinimidyl ester

RMSD:

root mean-square deviation

RP:

reverse-phase

TEA:

tetraethylammonium chloride

TFA:

trifluoroacetic acid

4AP:

4-aminopyridine

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Acknowledgments

This work is supported by the Russian Science Foundation (grant no. 14-14-00239). LSM710 microscope was granted by the M.V. Lomonosov Moscow State University Program of Development.

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Authors and Affiliations

  1. Biological Faculty, Lomonosov Moscow State University, Leninskie Gory 1, Moscow, 119992, Russia

    O. V. Nekrasova, A. D. Volyntseva, K. S. Kudryashova, V. N. Novoseletsky, E. A. Lyapina, K. V. Shaitan, M. P. Kirpichnikov & A. V. Feofanov

  2. Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, ul. Miklukho-Maklaya 16/10, 117997, Moscow, Russia

    O. V. Nekrasova, K. S. Kudryashova, A. V. Illarionova, S. A. Yakimov, Yu. V. Korolkova, M. P. Kirpichnikov & A. V. Feofanov

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  1. O. V. Nekrasova
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  2. A. D. Volyntseva
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  3. K. S. Kudryashova
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  4. V. N. Novoseletsky
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  5. E. A. Lyapina
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  6. A. V. Illarionova
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  7. S. A. Yakimov
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  9. K. V. Shaitan
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  10. M. P. Kirpichnikov
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  11. A. V. Feofanov
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Correspondence to A. V. Feofanov.

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Nekrasova, O.V., Volyntseva, A.D., Kudryashova, K.S. et al. Complexes of Peptide Blockers with Kv1.6 Pore Domain: Molecular Modeling and Studies with KcsA-Kv1.6 Channel. J Neuroimmune Pharmacol 12, 260–276 (2017). https://doi.org/10.1007/s11481-016-9710-9

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