Ignatova Anastasia A, Kryukova Elena V, Novoseletsky Valery N, Kazakov Oleg V, Orlov Nikita A, Korabeynikova Varvara N, Larina Maria V, Fradkov Arkady F, Yakimov Sergey A, Kirpichnikov Mikhail P, Feofanov Alexey V, Nekrasova Oksana V
Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Moscow 117997, Russia.
Department of Biology, Shenzhen MSU-BIT University, Shenzhen 518115, China.
Cells. 2024 Dec 18;13(24):2096. doi: 10.3390/cells13242096.
Advanced molecular probes are required to study the functional activity of the Kv1.2 potassium channel in normal and pathological conditions. To address this, a fully active Kv1.2 channel fused with fluorescent protein mKate2 (K-Kv1.2) was engineered that has high plasma membrane presentation due to the S371T substitution, and hongotoxin 1 (HgTx1) fused with eGFP at the C-terminus (HgTx-G) was produced. HgTx-G and HgTx1 N-terminally labeled with Atto488 fluorophore were shown to be fluorescent probes of Kv1.2 in cells with dissociation constants () of 120 and 80 pM, respectively. K-Kv1.2 and HgTx-G were used as components of an analytical system to study peptide blockers of the channel and helped to find out that Ce1 and Ce4 peptides from venom possess high affinity ( of 10 and 30 pM) and selectivity for Kv1.2. Using molecular docking and molecular modeling techniques, the complexes of Kv1.2 with HgTx1, Ce1, and Ce4 were modeled, and determinants of the high affinity binding were proposed. New fluorescent probes and selective blockers of Kv1.2 can be used to resolve Kv1.2-related challenges in neuroscience and neuropharmacology.
需要先进的分子探针来研究正常和病理条件下Kv1.2钾通道的功能活性。为了解决这个问题，构建了一种与荧光蛋白mKate2融合的完全活性Kv1.2通道（K-Kv1.2），由于S371T替换，其具有高细胞膜表达，并且制备了在C末端与eGFP融合的红藻毒素1（HgTx1）（HgTx-G）。HgTx-G和在N末端用Atto488荧光团标记的HgTx被证明是细胞中Kv1.2的荧光探针，其解离常数（）分别为120和80 pM。K-Kv1.2和HgTx-G用作分析系统的组成部分来研究该通道的肽阻滞剂，并有助于发现来自毒液的Ce1和Ce4肽对Kv1.2具有高亲和力（10和30 pM）和选择性。使用分子对接和分子建模技术，对Kv1.2与HgTx1、Ce1和Ce4的复合物进行建模，并提出了高亲和力结合的决定因素。新型Kv1.2荧光探针和选择性阻滞剂可用于解决神经科学和神经药理学中与Kv1.2相关的挑战。
